Method for Regulating Protein Function in Cells In Vivo Using Synthetic Small Molecules

ABSTRACT

Methods and compositions for the rapid and reversible destabilizing of specific proteins in vivo using cell-permeable, synthetic molecules are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/051,303 filed May 7, 2008, which is incorporated by reference herein.

STATEMENT REGARDING GOVERNMENT INTEREST

This work was supported in part by National Institutes of Health grants(GM068589 and GM073046). Accordingly, the United States government hascertain rights in this invention.

TECHNICAL FIELD

Methods and compositions for the rapid and reversible destabilizing ofspecific proteins in vivo using cell-permeable, synthetic molecules aredescribed.

BACKGROUND

Techniques that target gene function at the level of DNA and mRNAprovide powerful methods for modulating the expression of proteinsencoded by specific genes. For example, the tet/dox and Cre/lox systemshave been widely used to target gene expression at the transcriptionallevel (Ryding, A. D. S. et al. (2001) J. Endocrinol. 171:1-14) and RNAinterference is rapidly being adopted as a method to achievepost-transcriptional gene silencing (Fire, A. et al. (1998) Nature391:806-811; Medema, R. H. (2004) Biochem. J. 380:593-603; Raab, R. M.and Stephanopoulos, G. (2004) Biotechnology & Bioengineering88:121-132).

However, methods for regulating protein function directly are limited,especially in mammalian cells. Inhibitors or activators of particularproteins have been identified, and often take the form of cell-permeablesmall molecules. Many of these molecules have found widespread use asbiological probes, often because the speed, dosage-dependence, andreversibility of their activities, which complement methods forgenetically modulating gene expression (Schreiber, S. L. (2003) Chem. &Eng. News 81:51-61). However these inhibitors or activators are oftenpromiscuous, affecting several proteins rather than a specific protein(Davies, S. P. et al. (2000) Biochem. J. 351:95-105; Bain, J. et al.(2003) Biochem. J. 371:199-204; Godl, K. et al. (2003) Proc. Natl. Acad.Sci. U.S.A. 100:15434-15439; Tan, D. S. (2005) Nat. Chem. Biol. 1:74-84;Mayer, T. U. et al. (1999) Science, 286:971-974).

Shokat and coworkers have developed a method by which specific kinasescan be inhibited using a small-molecule modulator (Shah et al., 1997;Bishop, A. C. et al. (1998) Current Biology 8:257-266). This methodinvolves mutating the protein of interest, typically replacing a largeconserved residue in the active site with a smaller residue, such asglycine or alanine. Specificity is achieved by chemically modifying apromiscuous inhibitor to include a bulky side-chain substituent (e.g.,R-group), which fills the corresponding cavity in the binding site ofthe modified protein of interest, while preventing productiveinteractions with other kinases. While this so-called “bump-hole”approach has been successful both in cultured cells and in mice (Bishop,A. C. et al. (2000) Nature 407:395-401; Wang, H. et al., (2003) Proc.Natl. Acad. Sci. U.S.A. 100:4287-4292, Chen, X. et al. (2005) Neuron46:13-21), it appears to be limited to ATPases and GTPases. Additionalmethods are required to probe the function of a wider variety ofproteins.

Other investigators have devised alternative strategies to perturbprotein function by exploiting existing cellular processes (Banaszynski,L. A. et al. (2006) Chem. Biol. 13:11-21). For example, Varshavsky andcoworkers developed methods for controlling protein function based onthe importance of certain N-terminal residues for protein stability(Bachmair, A. et al. (1986). Science 234:179-186). Szostak and coworkersshowed that a small peptide sequence could be fused to the N-terminus ofa protein of interest to modulate protein stability (Park, E-C. et al.(1992) Proc. Natl. Acad. Sci. U.S.A. 89:1249-1252). Varshavsky andcoworkers have further isolated a temperature-sensitive peptide sequencethat greatly reduced the half-life or dihydrofolate reductase (DHFR) atthe non-permissive temperatures (Dohmen, R. J. et al. (1994) Science263:1273-1276). This approach has been used to study proteins in yeast(Labib, K. et al. (2000) Science 288:1643-1646; Kanemaki, M. et al.(2003) Nature 423:720-724). More recently, several researchers haveengineered systems in which dimeric small molecules are used toconditionally target fusion proteins for degradation via E3 ligase orthe proteasome, itself (Schneekloth et al., 2004; Janse, D. M. et al.(2004) J. Biol. Chem. 279:21415-21420). However, these systems requireeither a prior knowledge of the high-affinity ligands that modulate theactivity of a protein of interest or they are restricted to geneticallyengineered yeast strains.

An alternative approach for controlling protein function directly is tointerfere with subcellular localization. Several methods are availableto regulate protein localization using small-molecule by takingadvantage of the FKBP-rapamycin-FRB ternary complex (Kohler, J. J. andBertozzi, C. R. (2003) Chem. Biol. 10:1303-1331 and Inoue, T. et al.(2005) Nature Methods 2:415-418). Rapamycin and FK506 are potent,commercially available immunosuppressive agents, which are ligands ofthe FK506-binding protein (FKBP12, FKBP). Rapamycin also binds toFKBP-rapamycin-associated protein (FRAP). FRAP is also called themammalian target of rapamycin (mTOR), rapamycin and FKBP target 1(RAFT1), and FRB. Rapamycin binds to and inhibits mTOR by interactingwith its FKBP-rapamycin-binding (FRB) domain to inhibit/delay G1 cellcycle progression in mammalian cells (see, e.g., Choi, J. et al. (1996)Science 273:239-42 and Vilella-Bach, M. et al. (1999) J. Biol. Chem.274:4266-72. The FKBP-rapamycin-binding domain is required forFKBP-rapamycin-associated protein kinase activity and G1 progression.Fusions of proteins of interest can be made to either FKBP or to the FRPdomain of FRAP/mTOR. Colocalization of the protein of interest isinduced upon addition of rapamycin.

Because rapamycin has inherent biological activity, researchers havedeveloped a “bump-hole” strategy (similar to that employed by Shokat andcoworkers), wherein rapamycin derivatives possessing large substituentsat the FRB binding interface bind poorly to wild-type FRB and in turnthe biologically relevant target FRAP/mTOR, with binding restored uponintroduction of compensatory cavity-forming mutations in FRB.Specifically, a C20-methallyl-rapamycin derivative (MaRap) binds to atriple-mutated variant of FRB called FRB* (Liberles, S. D. et al. (1997)Proc. Natl. Acad. Sci. U.S.A. 94:7825-7830).

While these methods for regulating protein function directly arenoteworthy, there has yet to be described a convenient, general methodfor regulating protein function, particularly a method that does notrequire the interaction of multiple proteins. Improved methods forregulating protein function directly, particularly in mammalian cellsand in animals, are needed.

BRIEF SUMMARY

Compositions, systems, and methods for modulating the stability ofproteins in vitro and in vivo using cell-permeable small molecules aredescribed. Proteins of interest are fused to a stability-affectingprotein capable of interacting with a small-molecule ligand, thepresence, absence, or amount of which is used to modulate the stabilityof the fusion protein.

In one aspect, an in vivo conditional protein stability system isprovided comprising a nucleic acid sequence encoding a fusion proteinthat comprises a protein of interest fused to a single-polypeptidechain, ligand-dependent, stability-affecting protein derived from anaturally-occurring ligand binding protein, and a ligand that binds tothe stability-affecting protein to modulate stability of thestability-affecting protein, wherein upon introduction of the nucleicacid sequence to a eukaryotic cell the fusion protein is expressed andthe stability of the fusion protein can be modulated by administeringthe ligand to the eukaryotic cell.

In one embodiment, the single-polypeptide chain, ligand-dependent,stability-affecting protein is a FKBP variant protein. In anotherembodiment the naturally-occurring ligand binding protein is a naturallyoccurring or wildtype FKBP protein.

In some embodiments, the protein of interest is a reporter protein. Insome embodiments, the protein of interest is a therapeutic protein.

In one embodiment, the protein of interest is a cytokine. In yet anotherembodiment, the protein of interest is TNF-α or IL-2.

In one embodiment, the ligand is Shield1.

In one embodiment, the stability-affecting protein destabilizes theprotein of interest or fusion protein in the absence of a ligand. Inanother embodiment, the stability-affecting protein does not destabilizethe protein of interest in the presence of ligand. In yet anotherembodiment, the stability-affecting protein destabilizes the protein ofinterest or fusion protein in the absence of the ligand to a greaterdegree or extent than it destabilizes the protein of interest in thepresence of the ligand.

In one embodiment, the stability-affecting protein destabilizes theprotein of interest or fusion protein in the presence of a ligand. Inyet another embodiment, the stability-affecting protein does notdestabilize the protein of interest or fusion protein in the absence ofligand. In yet another embodiment, the stability-affecting proteindestabilizes the protein of interest or fusion protein in the presenceof the ligand to a greater degree or extent than it destabilizes theprotein of interest or fusion protein in the absence of the ligand.

In some embodiments, the stability-affecting protein destabilizes theprotein of interest by causing an increase in the degradation ordestruction of the protein of interest or the fusion protein when notbound to the ligand as compared to the level of degradation of theprotein of interest or the fusion protein when the stability-affectingprotein is bound to the ligand.

In one embodiment, the ligand binds preferably to the single-polypeptidechain, ligand-dependent, stability-affecting protein as compared to thenaturally-occurring ligand binding protein.

In one embodiment, the eukaryotic cells are transformed with the nucleicacid. In another embodiment, the eukaryotic cells are transformed withthe nucleic acid to produce stably transformed eukaryotic cells. Inanother embodiment, the transformed eukaryotic cells are implanted intoan animal. In yet another embodiment, the transformed eukaryotic cellsare implanted into immunodeficient mice as xenografts.

In one embodiment, the nucleic acid sequence is in a viral vector. Inanother embodiment, the viral vector is a pox virus. In yet anotherembodiment, the viral vector is a vaccinia virus. In yet anotherembodiment, the viral vector is a vvDD.

In one embodiment, the ligand is administered to the eukaryotic cells inculture. In another embodiment, the ligand is administered to the cellsby injecting the ligand into the animal intraperitoneally orintravenously.

In another aspect, a method for modulating stability of a protein ofinterest in vivo comprising introducing into a eukaryotic cell a nucleicacid comprising a polynucleotide which encodes a fusion protein whereinthe fusion protein comprises a protein of interest and asingle-polypeptide chain, ligand-dependent, stability-affecting proteinderived from a naturally-occurring ligand binding protein, andadministering a ligand to the eukaryotic cell is provided wherein theligand binds to the single-polypeptide chain, ligand-dependent,stability-affecting protein to modulate stability of the fusion protein.

In one embodiment the single-polypeptide chain, ligand-dependent,stability-affecting protein is a FKBP variant protein. In anotherembodiment the naturally-occurring ligand binding protein is a naturallyoccurring or wildtype FKBP protein.

In some embodiments, the protein of interest is a reporter protein. Insome embodiments, the protein of interest is a therapeutic protein.

In one embodiment, the protein of interest is a cytokine. In yet anotherembodiment, the protein of interest is TNF-α or IL-2.

In one embodiment, the ligand is Shield1.

In one embodiment, the stability-affecting protein destabilizes theprotein of interest or fusion protein in the absence of a ligand. Inanother embodiment, the stability-affecting protein does not destabilizethe protein of interest or fusion protein in the presence of ligand. Inyet another embodiment, the stability-affecting protein destabilizes theprotein of interest or fusion protein in the absence of the ligand to agreater degree or extent than it destabilizes the protein of interest orfusion protein in the presence of the ligand.

In yet another embodiment, the stability-affecting protein destabilizesthe protein of interest or fusion protein in the presence of a ligand.In yet another embodiment, the stability-affecting protein does notdestabilize the protein of interest or fusion protein in the absence ofligand. In yet another embodiment, the stability-affecting proteindestabilizes the protein of interest or fusion protein in the presenceof the ligand to a greater degree or extent than it destabilizes theprotein of interest or fusion protein in the absence of the ligand.

In one embodiment, the ligand binds preferably to the single-polypeptidechain, ligand-dependent, stability-affecting protein as compared to thenaturally-occurring ligand binding protein.

In one embodiment, the nucleic acid sequence is introduced into theeukaryotic cell by transforming the eukaryotic cell in culture with aplasmid comprising the nucleic acid to produce stably transformedeukaryotic cells. In another embodiment, the stably transformedeukaryotic cells are administered to an animal. In yet anotherembodiment, the stably transformed eukaryotic cells are transplantedinto a mouse. In yet another embodiment, the stably transformedeukaryotic cells are transplanted as a xenograft into an immunodeficientmouse.

In one embodiment, the nucleic acid sequence is introduced into theeukaryotic cell by transforming the eukaryotic cell in culture with aviral vector which comprises the nucleic acid sequence. In anotherembodiment, the nucleic acid is introduced into the eukaryotic cell byadministering to an animal a viral vector which comprises the nucleicacid sequence. In yet another embodiment, the animal has tumor cells.

In another aspect, a method for modulating cellular proliferation in ananimal, comprising administering to the animal a nucleic acid comprisinga polynucleotide which encodes a fusion protein wherein the fusionprotein comprises a protein of interest and a single-polypeptide chain,ligand-dependent, stability-affecting protein derived from anaturally-occurring ligand binding protein, and administering a ligandwhich binds preferably to the single-polypeptide chain,ligand-dependent, stability-affecting protein as compared to thenaturally-occurring ligand binding protein is provided.

In one embodiment the single-polypeptide chain, ligand-dependent,stability-affecting protein is a FKBP variant protein. In anotherembodiment the naturally-occurring ligand binding protein is a naturallyoccurring or wildtype FKBP protein.

In some embodiments, the protein of interest is a reporter protein. Insome embodiments, the protein of interest is a therapeutic protein.

In one embodiment, the protein of interest is a cytokine. In yet anotherembodiment, the protein of interest is TNF-α or IL-2.

In one embodiment, the ligand is Shield1.

In one embodiment, the stability-affecting protein destabilizes theprotein of interest in the absence of a ligand. In another embodiment,the stability-affecting protein does not destabilize the protein ofinterest in the presence of ligand. In yet another embodiment, thestability-affecting protein destabilizes the protein of interest orfusion protein in the absence of the ligand to a greater degree orextent than it destabilizes the protein of interest or fusion protein inthe presence of the ligand.

In yet another embodiment, the stability-affecting protein destabilizesthe protein of interest in the presence of a ligand. In yet anotherembodiment, the stability-affecting protein does not destabilize theprotein of interest in the absence of ligand. In yet another embodiment,the stability-affecting protein destabilizes the protein of interest orfusion protein in the presence of the ligand to a greater degree orextent than it destabilizes the protein of interest or fusion protein inthe absence of the ligand.

In one embodiment, the ligand binds preferably to the single-polypeptidechain, ligand-dependent, stability-affecting protein as compared to thenaturally-occurring ligand binding protein.

In one embodiment, the method of administering the nucleic acid to theanimal comprises transforming a eukaryotic cell with the nucleic acid toproduce a stably transformed eukaryotic cell and implanting the stablytransformed eukaryotic cell into the animal.

In one embodiment, the eukaryotic cells are tumor cells andadministering the ligand to the animal modulates establishment,progression, and/or growth of the tumor.

In another embodiment, administering the ligand to the animal inhibitstumor growth. In another embodiment, administering the ligand improvessurvival of the animal.

In one embodiment, the animal has tumor cells and administering thenucleic acid to the animal comprises administering a viral vector orvirus to the animal, wherein the viral vector or virus harbors thenucleic acid. In another embodiment, the viral vector or virus is a poxvirus. In yet another embodiment, the viral vector or virus is avaccinia virus. In yet another embodiment, the viral vector or virus isa vvDD virus.

In another aspect, a method for modulating tumor establishment, tumorprogression and/or tumor growth in an animal is provided, comprisingintroducing into cells of the animal a nucleic acid sequence encoding afusion protein comprising a therapeutic protein and a single-polypeptidechain, ligand-dependent, stability-affecting protein, and controllingexpression of the therapeutic protein by administering a preselectedamount of the ligand, is provided wherein the ligand binds to thestability-affecting protein to modulate the stability of the therapeuticprotein and the therapeutic protein modulate tumor progression ordevelopment.

In some embodiments, the nucleic acid sequence is in a plasmid. In someembodiments, the nucleic acid sequence is in a viral vector.

In some embodiments, the stability-affecting protein destabilizes theprotein of interest in the absence of the ligand. In another embodiment,the stability-affecting protein does not destabilize the protein in thepresence of the ligand.

In some embodiments, the ligand preferentially binds to thestability-affecting protein compared to the correspondingnaturally-occurring ligand-binding protein.

In some embodiments, the stability-affecting protein is a variant FKBPprotein and the fusion protein is stabilized in the presence of a FKBPligand. In particular embodiments, the FKBP ligand is Shield1.

In some embodiments, the therapeutic protein is a cytokine. Inparticular embodiments, the therapeutic protein is interleukin-2.

In one embodiment, the ligand is administered by intraperitoneal orintravenous injection of the ligand into animal.

In some embodiments, the therapeutic protein modulates tumorestablishment. In some embodiments, the therapeutic protein modulatestumor progression. In some embodiments, the therapeutic protein preventstumor establishment or progression. In some embodiments, the therapeuticprotein prevents or inhibits tumor growth.

In one aspect, an in vivo conditional protein stability system isprovided, comprising a nucleic acid sequence encoding a fusion proteinthat includes a protein of interest fused in-frame to asingle-polypeptide chain, ligand-dependent, stability-affecting proteinderived from a naturally-occurring ligand binding protein, and a ligandthat binds to the stability-affecting protein to modulate its stability,wherein upon introduction of the nucleic acid sequence to cells of anorganism the fusion protein is expressed and the stability of the fusionprotein can be modulated by amount of ligand present in the cells of theorganism.

In some embodiments, the stability-affecting protein destabilizes theprotein of interest in the absence of the ligand and does notdestabilize the protein in the presence of the ligand. In someembodiments, the stability-affect protein destabilizes the protein ofinterest in the absence of the ligand to a greater degree or extent thanin the presence of the ligand.

In some embodiments, the stability-affecting protein does notdestabilize the protein of interest in the absence of the ligand anddestabilizes the protein in the presence of the ligand. In someembodiments, the stability-affect protein destabilizes the protein ofinterest in the presence of the ligand to a greater degree or extentthan in the absence of the ligand.

In some embodiments, the ligand preferentially binds to thestability-affecting protein compared to the naturally-occurringligand-binding protein.

In some embodiments, the stability-affecting protein is a variant FKBPprotein and the fusion protein is stabilized in the presence of a FKBPligand. In particular embodiments, the FKBP ligand is Shield1.

In some embodiments, the protein of interest is a reporter protein. Insome embodiments, the protein of interest is a therapeutic protein.

In a related aspect, a method of using the described in vivo conditionalprotein stability system for controlling the expression of protein isprovided. In some embodiments, the protein of interest is a reporterprotein or a therapeutic protein.

In another aspect, a method for modulating tumor establishment orprogression in an animal is provided, comprising introducing into cellsof the animal a nucleic acid sequence encoding a fusion protein thatincludes a therapeutic protein fused in-frame to a single-polypeptidechain, ligand-dependent, stability-affecting protein, and controllingexpression of the therapeutic protein by administering a preselectedamount of the ligand, wherein the ligand binds to thestability-affecting protein to modulate the stability of the therapeuticprotein and the therapeutic protein modulate tumor progression ordevelopment.

In some embodiments, the nucleic acid sequence is in a plasmid. In someembodiments, the nucleic acid sequence is in a viral vector.

In some embodiments, the stability-affecting protein destabilizes theprotein of interest in the absence of the ligand and stabilizes theprotein in the presence of the ligand.

In some embodiments, the ligand preferentially binds to thestability-affecting protein compared to the correspondingnaturally-occurring ligand-binding protein.

In some embodiments, the stability-affecting protein is a variant FKBPprotein and the fusion protein is stabilized in the presence of a FKBPligand. In particular embodiments, the FKBP ligand is Shield1.

In some embodiments, the therapeutic protein is a cytokine. Inparticular embodiments, the therapeutic protein is interleukin-2.

In some embodiments, the therapeutic protein modulates tumorestablishment. In some embodiments, the therapeutic protein modulatestumor progression. In some embodiments, the therapeutic protein preventstumor establishment or progression.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are diagrams illustrating a method for conditionallycontrolling protein stability. (FIG. 1A) A genetic fusion of adestabilizing domain (DD) to a protein of interest (POI) results indegradation of the entire fusion protein. Addition of a ligand for thedestabilizing domain protects the fusion protein from degradation. (FIG.1B) Synthetic ligands SLF* and Shield1 for FKBP F36V. FIG. 1C is aschematic illustrating the screening strategy described in the text.

FIGS. 2A-2D are graphs showing the results of experiments tocharacterize FKBP variants that display Shield1-dependent stability. Thedata for FIGS. 2A-2D are presented as the average mean fluorescenceintensity (MFI)±SEM relative to that of the maximum fluorescenceintensity observed for the individual mutant. Experiments were performedin triplicate. (FIG. 2A) Fluorescence of FKBP-YFP fusion proteinsexpressed in NIH3T3 cells as determined by flow cytometry in the absenceof Shield1. (FIG. 2B) NIH3T3 cells stably expressing FKBP-YFP fusionproteins were treated with three-fold dilutions of Shield1 (1 μM to 0.1nM) and the fluorescence was monitored by flow cytometry. (FIG. 2C)NIH3T3 cells stably expressing FKBP-YFP fusion proteins were eithermock-treated (circles) or treated with 30 nM Shield1 (squares), 100 nMShield1 (diamonds), 300 nM Shield1 (crosses), or 1 μM Shield1(triangles). Fluorescence was monitored over time using flow cytometry.MFI was normalized to 100% of cells treated with 1 μM Shield1 at 24 hrs.(FIG. 2D) NIH3T3 cells stably expressing FKBP-YFP fusion proteins weretreated with 1 μM Shield1 for 24 hours at which point the cells werewashed with media to remove Shield1, and the decrease in fluorescencewas monitored using flow cytometry.

FIGS. 2E-2G show the results of immunoblot analysis to characterize FKBPvariants that display Shield1-dependent stability. (FIG. 2E) FKBP-YFPfusion proteins were either mock-treated or treated with 1 μM Shield1for 24 hrs then subjected to immunoblot analysis with an anti-FKBPantibody. (FIG. 2F) NIH3T3 cells stably expressing F15S-YFP andL106P-YFP were treated with 1 μM Shield1 for 24 hrs. The cells were thenwashed with media and treated with 10 μM MG132 in the presence orabsence of 1 μM Shield1 for 4 hrs. Immunoblot analysis was performedusing an anti-YFP antibody. (FIG. 2G) HeLa cells were transfected withsiRNA against lamin A/C and monitored over time. The time required forthe reduction of lamin A/C was compared to the time required fordegradation of L106P-YFP upon removal of Shield1 from NIH3T3 cellsstably expressing the fusion protein.

FIG. 3 is a graph showing the reversible nature of small-moleculeregulation of intracellular protein levels. A population of NIH3T3 cellsstably expressing the L106P-YFP fusion protein was treated withdifferent concentrations of Shield1 over the course of one week, andsamples of the population were assayed for fluorescence by flowcytometry at the indicated time points. Predicted fluorescence is basedon the dose-response experiment shown in FIG. 2B.

FIGS. 4A and 4B show the results of immunoblot analysis demonstratingthat the FKBP destabilizing domain confers Shield 1-dependent stabilityto a variety of different proteins. (FIG. 4A) FKBP variants F15S andL106P were fused to the N-termini of several different proteins andtransduced into NIH3T3 cells. Cell populations stably expressing thefusions were then either mock-treated or treated with 1 μM Shield1, andcell lysates were subjected to immunoblot analysis using antibodiesspecific for the protein of interest. Endogenous proteins served asloading controls (when detected) and Hsp90 served as a loading controlotherwise. (FIG. 4B) FKBP variants D100G and L106P were fused to theC-termini of several different proteins of interest and treated asabove.

FIG. 5 is a graph showing that the destabilizing domain confersShield1-dependent stability to a transmembrane protein. FKBP variantsD100G and L106P were fused to the C-terminus of CD8α. NIH3T3 cellsstably expressing the fusion proteins were divided into three pools(groups). The first group (−) was mock-treated, the second group (+) wastreated with 1 μM Shield1 for 24 hrs, and the third group (+/−) wastreated with 1 μM Shield1 for 24 hrs, and then washed with media andcultured for 24 hr in the absence of Shield1. Live cells were thenprobed with a FITC-conjugated anti-CD8α antibody and assayed by flowcytometry. Data are presented as the average mean fluorescence intensity±SEM from an experiment performed in triplicate.

FIG. 6A shows the results of an experiment using a “drug-OFF” system, inwhich a FKBP protein attached to a diversity element sequence was fusedto YFP. YFP fluorescence could be detected by FACS analysis in theabsence of the ligand (Shield1) but was almost undetectable in thepresence of the ligand (n=5 clones).

FIGS. 6B and 6C shows the results of an experiment performed inToxoplasma. The fluorescence of parasitophorous vacuoles in infectedcells was monitored by time lapse microscopy following addition (FIG.6B) or removal of ligand (FIG. 6C). Background fluorescence isindicated.

FIGS. 6D and 6E shows the results of an experiment performed in aPlasmodium. An antibody to YFP protein was used to detect fusionproteins in transfected cells, which are greatly increased in thepresence of ligand.

FIGS. 7A and 7B are graphs showing the results of an experiment in whichSCID mice were challenged with tumor cells genetically manipulated toexpress luciferase fused to a stability-affecting protein.

FIG. 8 is a graph showing the kinetics of FKBP-YFP fluorescence in cellsupon addition of Shield1. NIH3T3 cells stably expressing the indicatedFKBP-YFP fusions were treated with 1 μM Shield1, and increases influorescence were monitored over time using flow cytometry. Data arepresented as the average mean fluorescence intensity (MFI) ±SEM relativeto that of the maximum fluorescence intensity observed for theindividual variant. The experiment was performed in triplicate, and MFIwas normalized to 100% at 24 hr.

FIG. 9 shows the results of immunoblot analysis demonstrating thatdegradation of FKBP-YFP fusions is mediated by the proteosome. NIH3T3cells stably expressing F15S-YFP and L106P-YFP were treated with 1 μMShield1 for 24 hrs. The cells were then washed and treated with 10 μMlactacystin in the presence and absence of 1 μM Shield1 for 4 hrs.Immunoblot analysis was performed using an anti-YFP antibody.

FIG. 10 shows the results of immunoblot analysis comparing the kineticsof destruction-domain-mediated degradation to the kinetics ofRNAi-mediated silencing. HeLa cells were transfected with 30 nM siRNAagainst lamin A/C and monitored over time. The time required for thereduction in levels of lamin A/C was compared to the time required forthe degradation of L106P-YFP upon removal of Shield1 from the cellsstably expressing the fusion protein.

FIG. 11 is a graph showing the levels of fluorescence of variousYFP-FKBP fusion proteins (i.e., C-terminal FKBP mutants) in response todifferent concentrations of Shield1. NIH3T3 cells stably expressingYFP-FKBP fusion proteins were incubated with three-fold dilutions ofShield1 (3 μM to 0.1 nM) and fluorescence was monitored by flowcytometry. The data are presented as MFI ±SEM relative to that of themaximum fluorescence intensity observed for the individual variant. Theexperiment was performed in triplicate.

FIG. 12 is a graph showing the kinetics of decay of YFP-FKBPfluorescence upon removal of Shield1. NIH3T3 cells stably expressingYFP-FKBP fusion proteins were treated with 1 μM Shield1 for 24 hours, atwhich point the cells were washed with media to remove Shield 1. Thedecrease in fluorescence was monitored using flow cytometry. Data arepresented as the average mean fluorescence intensity relative to that ofthe maximum fluorescence intensity observed for the individual mutant.

FIG. 13 is a graph showing that FKBP-YFP fusion proteins are stabilizedby multiple FKBP ligands. NIH3T3 cells stably expressing the L106P-YFPfusion were treated with three-fold dilutions of FK506 (30 μM to 10 nM)or Shield1 (3 μM to 0.1 nM) and fluorescence was monitored by flowcytometry. Data are presented as the average mean fluorescence intensity±SEM relative to that of the maximum fluorescence intensity observed forthe L106P-YFP mutant. Experiments were performed in triplicate.

FIG. 14 is a graph showing fluorescence levels obtained using YFP-DHFRand YFP-DHFR fusion proteins. YFP fused to wild-type, G121V, and Y100Iversions of DHFR were subjected to fluorescence analyses in the presenceof 10 μM or 100 μM TMP.

FIG. 15 is a graph showing fluorescence levels in NIH3T3 cells stablyexpressing DHFR-YFP fusion proteins and treated with ten-fold dilutionsof TMP (10 μM to 1 nM), or mock-treated with DMSO. The fluorescencelevels were monitored by flow cytometry. MFI was normalized to 100% ofcells treated with 10 μM TMP at 24 hrs.

FIGS. 16A and 16B are graphs showing YFP fluorescence levels in NIH3T3cells expressing YFP-DHFR fusion proteins and treated with ten-folddilutions of TMP, or mock-treated with DMSO. (FIG. 16A) Y100I and G121Vmutants. (FIG. 16B) F103L and N18T/A19V mutants.

FIG. 17A is a graph showing a comparison of the stability of YFP fusedat the N-terminus of several DHFR mutants. FIG. 17B shows the results ofan immunoblot experiment using an antibody specific for YFP in thepresence and absence of ligand.

FIG. 18A is a graph showing the kinetics of YFP-DHFR decay followingwithdrawal of ligand. FIG. 18B is a graph showing the kinetics ofYFP-DHFR stabilization following addition of ligand.

FIG. 19A is a graph showing the relative mean fluorescence intensity incells harboring mutant DHFR-YFP fusion proteins in the absence andpresence of ligand. FIG. 19B shows the results of a dose responseexperiment, in which the cells expressing DHFR-YFP mutants were exposedto increasing amounts of ligand.

FIGS. 20A and 20B show the kinetics of decay and stabilization,respectively, of three DHFR mutants fused to the N-terminus of YFP.

FIGS. 21A-21D show the results of experiments showing conditionalregulation of protein stability in vivo. SCID mice bearing HCT116L106P-tsLuc xenografts (50-100 mm³) were either untreated (top row) ortreated i.p. with Shield1 (10 mg/kg, bottom row) and bioluminescentsignals were imaged over time (FIG. 21A). FIG. 21B shows quantificationof the results from FIG. 21A. Mice were treated i.p. with Shield1 at 3mg/kg (diamonds), 6 mg/kg (inverted triangles), or 10 mg/kg (triangles)and imaged over time (FIG. 21C). Mice bearing HCT116-tsLuc xenograftswere either untreated (squares) or treated with Shield1 (10 mg/kg,triangles) every 48 hr and imaged over time (FIG. 21D). Data for FIG.21B-21 D are presented as the average bioluminescence detected withinregions of interest drawn around the tumors ±SEM (n=4 to 10).

FIGS. 22A-22D show graphs relating to the conditional stabilization of asecreted immunomodulatory protein leads. FIG. 22A shows a graphresulting from an experiment in which HCT116 L-L106P-IL-2 cells weretreated with various concentrations of Shield1 and culture media wasassayed for the presence of IL-2. Data are represented as the averageIL-2 concentration ±SEM (n=3). FIG. 22B shows a graph resulting from anexperiment in which CD1 nu-/nu- mice bearing subcutaneous HCT116L-L106P-IL-2 tumors were either untreated (diamonds) or treated i.p.with Shield1 at 5 mg/kg (triangles) or 10 mg/kg (inverted triangles)every 48 hr beginning 5 days post-transplantation (arrow).Alternatively, mice received HCT116 L-L106P-IL-2 cells that had beenpre-treated with 1 μM Shield1 for 24 hr, and were then treated withShield1 (10 mg/kg) every 48 hr beginning on day 0 (squares). Tumorvolume was determined by caliper measurement and monitored over time.Data are represented as the average tumor volume ±SEM (n=5). At Day 16,all Shield1 treated groups displayed significantly reduced tumor burdenrelative to controls (p=0.0019 for 10 mg/kg; 0.0002 for 5 mg/kg and0.0046 for pre-treat group). FIG. 22C shows a graph resulting from anexperiment in which tumors from mice treated with Shield1post-transplantation were collected 48 hr after the start of Shield1treatment, weighed ex vivo, homogenized, and the concentration of IL-2per gram tumor tissue determined by ELISA (n=4). Tumors from micetreated with Shield1 at 10 mg/kg contained significantly higher levelsof IL-2 than tumors from mice treated at 5 mg/kg (p=0.032), which inturn produced more IL-2 than tumors from untreated mice (p=0.0028). FIG.22D shows a graph resulting from an experiment in which mice treatedwith Shield1 post-transplantation were bled 48 hr after the start ofShield1 treatment, and the concentration of IL-2 in the serum wasdetermined by ELISA. Shield1 treatment at 10 mg/kg (n=7) producedsignificantly higher levels of serum IL-2 relative to control mice (n=6,p=0.0004), whereas treatment at 5 mg/kg (n=6) did not produce anyincrease in serum IL-2 levels.

FIGS. 23A-23C relate to the systemic, targeted-delivery of aconditionally stabilized protein. FIG. 23A shows a graph resulting froman experiment in which HCT116 cells were infected with vvDD L106P-tsLucand then mock treated (squares) or treated with Shield1 at 1 μM(triangles), 100 nM (inverted triangles), or 10 nM (diamonds). Data arerepresented as the average luminescence ±SEM (n=3). FIG. 23B shows agraph resulting from an experiment in which SCID mice bearingsubcutaneous HCT116 xenografts (50-100 mm3) received a single tail veininjection of vvDD L106P-tsLuc (1×10⁸ PFU/mouse), and after 72 hours wereeither untreated (C, control) or treated with Shield1 (10 mg/kg, mice1-4). Bioluminescent signals were imaged over time. FIG. 23C is a graphshowing quantification of the bioluminescent signals produced from theindicated regions of interest around the tumors shown in FIG. 23B. Datapresented are the average bioluminescence ±SEM (n=4).

FIGS. 24A and 24B show the antitumor effects of conditionalstabilization of tumor necrosis factor delivered via a vvDD virus. Viralload within the tumor was assayed by measuring constitutive viral geneexpression (bioluminescence) for each group at the indicated time pointsafter vvDD treatment. As shown in FIG. 24A, Shield1 treatment starting72 hr after vvDD administration (vvDD+Shield1) resulted in significantlygreater levels of viral gene expression in the tumor than when Shield1treatment was started prior to vvDD administration (vvDD Pre-Shield1;p=0.035 at 2 days; p=0.035 at 4 days and p=0.002 at 7 days). FIG. 24Bshows a Kaplan-Meier survival graph relating to mice treated withShield1 and vvDD in various combinations.

FIG. 25 relates to the systemic, targeted-delivery of a conditionallystabilized IL-2 fused to the L106P variant. The graph shows results froman experiment in which SCID mice bearing subcutaneous HCT116 xenografts(50-100 mm³) received a single tail vein injection of vvDD L-L106P-IL-2(1×10⁸ PFU/mouse), and after 72 hours were either untreated (indicatedby “No IL2”), treated with Shield1 prior to administration of the vvDDL-L106P-IL-2 construct (indicated by “Constitutive IL2”), or treatedwith Shield172 hours after administration of the vvDD L-L106P-IL-2construct (indicated by “IL2 after 3 days”).

FIG. 26 shows a Kaplan-Meier survival graph relating to mice treatedwith Shield1 and vvDD constructs. Squares represent mice treated withShield1 prior to administration with the vvDD L-L106D-IL-2 construct.Diamonds represent mice treated with the vvDD L-L106P-IL-2 constructprior to administration of Shield1. Triangles represent mice treatedwith the vvDD L-L106P-IL-2 construct only. Inverted triangles representmice treated with Shield1 only. Circles represent mice treated with PBS(phosphate buffered saline) only.

FIG. 27 shows infiltration of Treg cells into tumor cells of SCID micebearing subcutaneous HCT116 xenografts. Tumors were excised post-mortemand cells dissociated into a single cell suspension by grinding tissuethrough a cell filter. The single cell suspension was stained usingantibodies for T-reg markers (CD4+CD25+FoxP3+), and the percentage ofcells stained positive was determined by flow cytometry. Squaresrepresent mice treated with Shield1 prior to administration with thevvDD L-L106D-IL-2 construct. Diamonds represent mice treated with thevvDD L-L106P-IL-2 construct prior to administration of Shield1.Triangles represent mice treated with the vvDD L-L106P-IL-2 constructonly. Inverted triangles represent mice treated with Shield1 only.Circles represent mice treated with PBS (phosphate buffered saline)only.

FIG. 28 shows effects of NK-92 cells infected with the vvDD-L-L106P-IL2virus

FIG. 29 shows a list of sequences referred to in the application.

DETAILED DESCRIPTION

Any methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the teachings herein.All publications mentioned herein are incorporated herein by referencein their entirety for the purpose of describing and disclosing themethodologies which are reported in the publications which might be usedin connection with the teachings herein.

1. Definitions

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise.

As used herein, a “protein of interest” or “POI” is any protein, orfunctional fragment or derivative, thereof, that one skilled in the artwishes to study.

As used herein, “preferentially binds” means to bind with greaterefficiency to a subject molecule (such as a particular amino acidsequence) than another molecule. The difference in binding efficiencymay be 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 10,000fold, or more.

As used herein, “introduction of nucleic to cells” means transfection,transduction (infection), or transformation of nucleic acids (e.g., DNA)into cells, such that the nucleic acids may be used by the cell toexpress a protein of interest.

As used herein, “degradation” or “destruction” of a protein means itshydrolysis into smaller proteins or amino acids, such as by the cellularproteosome.

“Rapamycin” is a naturally-occurring, small molecule immunosuppressantthat is a ligand for FKBP.

“FKBP12” or “FKBP” is a 12 kDa protein that binds to the small-moleculesrapamycin and FK506. The rapamycin-FKBP complexes can bind to the FRBdomain of FRAP.

“FRAP,” “mTOR,” or “FRAP/mTOR” is a protein that binds, via its FRBdomain, to rapamycin, or the FKBP-rapamycin complex.

A “FKBP variant” refers to a protein wherein one or more amino acidresidues, e.g., at positions 15, 24, 25, 36, 60, 100, and 106, aresubstituted for an amino acid other than the amino acid in the FKBP F36Vprotein (SEQ ID NO: 1). Other amino acid positions that can besubstituted are indicated in the Tables and Figures.

“FRB*” is a FRB variant with three point mutations, i.e.,K2095P/T2098LN2101F (using mTOR numbering), designed to bind rapamycinanalogs such as MaRAP. Amino acid substitutions, or point mutations, aredenoted herein, and in accord with conventional practice, as the old(original) residue in single-letter code, followed by its codonlocation, followed by the substitute (mutant) amino acid insingle-letter code. For example, the F36V mutant of FKBP has Val inplace of Phe at position 36 of the FKBP protein.

“MaRAP” is C20-methallylrapamycin, a synthetic rapamycin derivative thatbinds FRB* but not FRB.

“Shield1” is a synthetic small molecule that binds to wild-type FKBP, aFKBP variant having a F36V mutation/substitution, and likely other FKBPvariants. Binding is about 1,000-fold tighter to the F36V variantcompared to wild-type FKBP (Clackson, T. et al. (1998) Proc. Natl. Acad.Sci. U.S.A. 95:10437-10442).

As used herein, a “single-protein, ligand-dependent destabilizationprotein,” “single-polypeptide chain, ligand-dependent,stability-affecting protein”, “single protein, stability-affectingprotein,” or “stability-affecting protein” is a single polypeptide thatfunctions as a ligand-dependent destabilization protein, as describedherein. Such a destabilizing protein does not require the formation of aternary complex, as is the case with the FKBP-rapamycin-FRB complex. Aparticular species is a “single-domain,” ligand-dependentdestabilization protein, wherein the single polypeptide comprises only asingle domain (i.e., folded structure or functional unit as determinedby X-ray crystallography, protease digestion, computer modeling, etc.

As used herein, “fused” means arranged in-frame as part of the samecontiguous sequence of amino acids in a polypeptide. Fusion can bedirect such there are no additional amino acid residues or via a linkerto improve performance or add functionality.

As used herein, “conservative amino acid substitutions” aresubstitutions that do not result in a significant change in the activityor tertiary structure of a selected polypeptide or protein. Suchsubstitutions typically involve replacing a selected amino acid residuewith a different residue having similar physico-chemical properties. Forexample, substitution of Glu for Asp is considered a conservativesubstitution since both are similarly-sized negatively-charged aminoacids. Groupings of amino acids by physico-chemical properties are knownto those of skill in the art.

As used herein, the terms “domain” and “region” are used interchangeablyherein and refer to a contiguous sequence of amino acids within a POI ordestabilizing domain, typically characterized by being either conservedor variable and having a defined function, such as ligand binding,conferring stability or instability, enzymatic function, etc.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably and without distinction to refer to a compound made upof a chain of amino acid residues linked by peptide bonds. Unlessotherwise indicated, the sequence for peptides is given in the orderfrom the “N” (or amino) terminus to the “C” (or carboxyl) terminus. Itis understood that polypeptides include a contiguous sequence of aminoacid residues.

A peptide or peptide fragment is “derived from” a parent peptide orpolypeptide if it has an amino acid sequence that is homologous to, butnot identical to, the parent peptide or polypeptide, or of a conservedfragment from the parent peptide or polypeptide.

Two amino acid sequences or two nucleotide sequences are considered“homologous” if they have an alignment score of >5 (in standarddeviation units) using the program ALIGN with the mutation gap matrixand a gap penalty of 6 or greater (Dayhoff, M. O., in Atlas of ProteinSequence and Structure (1972) Vol. 5, National Biomedical ResearchFoundation, pp. 101-110, and Supplement 2 to this volume, pp. 1-10.) Thetwo sequences (or parts thereof) are more preferably homologous if theiramino acids are greater than or equal to 50%, 70%, 80%, 90%, 95%, oreven 98% identical when optimally aligned using the ALIGN programmentioned above.

“Modulate” intends a lessening, an increase, or some other measurablechange, e.g., in the stability or biological function of a protein.

A “small molecule ligand” is a discrete small-molecule, well known inthe pharmaceutical and material sciences, which is to be distinguishedfrom, e.g., a polypeptide or nucleic acid, which is a polymer consistingof monomeric subunits. Small molecule ligands may be naturally-occurringor synthetic as exemplified by pharmaceutical products, laboratoryreagents, and the like.

“ddVV” refers to a Vaccinia virus having a double-deletion as describedin Thorne et al. (2006) Science, 311:1780-1784; McCart et al. (2001)Cancer Res. 61:8751-8757.

As used herein, a “variant” protein is a protein having an amino acidsequence that does not occur in nature, as exemplified by sequences inGenBank.

As used herein, a “mutant” is a mutated protein designed or engineeredto alter properties or functions relating to protein stabilizationand/or ligand binding.

2. Overview

The present composition, system, and method (generally, “system”) relateto the conditional stabilization of a protein of interest (POI) fused toa single-polypeptide chain, ligand-dependent, stability-affectingprotein in vivo. The stability-affecting protein, also referred to as aligand-binding domain, can be preselected to confer either stability orinstability to the entire fusion protein, depending on the presence orabsence of the ligand.

A feature of the conditional protein stability system is that thestability-affecting protein is of a “single ligand-single domain” type,which minimizes the number of components in the system. The system isillustrated using two different ligand-binding domains, namely, theFK506-binding protein (FKBP) and dihydrofolate reductase (DHFR), bindingdomains, in combination with appropriate ligands. Experiments performedin support of the systems are described below, along with embodimentsand examples of the system.

3. Stability-affecting protein derived from FKBP

a. Introduction

Rapamycin and FK506 are commercially available, cell-membrane permeable,FDA-approved immunosuppressive agents, which are ligands of theFK506-binding protein (FKBP12 or FKBP). The FKBP-rapamycin complex bindsto the FKBP-rapamycin-binding (FRB) domain of theFKBP-rapamycin-associated protein (FRAP). FRAP is also known as themammalian target of rapamycin (mTOR), rapamycin and FKBP target 1(RAFT1), and sometimes FRB. Rapamycin complexed with FKBP binds to andinhibits FRAP/mTOR at its FRB domain, which eventually inhibits/delayscell cycle progression through G₁ (see, e.g., Choi, J. et al. (1996)Science 273:239-42 and Vilella-Bach, M. et al. (1999) J. Biol. Chem.274:4266-72. Fusion polypeptides may be made between a proteins ofinterest (POI) and either FKBP and/or the FRB domain of FRAP/mTOR.Colocalization of the protein(s) of interest is induced upon addition ofrapamycin.

Because rapamycin has inherent biological activity, researchersdeveloped a “bump-hole” strategy (similar to that employed by Shokat andcoworkers), wherein rapamycin derivatives with bulky side-chainsubstituents would bind poorly to the FRB domain of awild-type/naturally-occurring FRAP/mTOR. Binding was restored byintroducing compensatory hole/cavity-forming mutations in the FRBdomain. In particular, the bulky side chain of a C20-methallyl-rapamycinderivative (MaRap) is accommodated by a triple-substituted variant ofFRB called FRB* (Liberles, S. D. et al. (1997) Proc. Natl. Acad. Sci.U.S.A. 94:7825-7830).

In fusing the FRB and FRB* domains to a kinase GSK-3β it was discoveredthat the levels of the GSK-3β-FRB* fusion protein were substantiallydecreased compared to an otherwise identical GSK-3-β-FRB fusion protein.The levels of the FRB* fusion protein were rescued (i.e., increased tolevels similar to those of the FRB fusion protein) upon the addition ofMaRap (Stankunas, K. et al. (2003) Mol. Cell. 12:1615-1624). FRB*appeared to confer conditional instability to multiple differentproteins in the absence of MaRap, with stabilization being dependent onthe interaction of two proteins (i.e., FKBP and FRB) via a smallmolecule (MaRap) that is expensive, difficult to synthesize andformulate, and exhibits poor pharmacokinetic properties in vivo.

b. Drug-ON System

The 107-residue FK506 and rapamycin-binding protein (FKBP) was selectedfor use as a destabilizing domain in a “drug-ON” system. FKBP has beenwidely studied, often in the context of fusion proteins, and numeroushigh-affinity ligands for FKBP have been developed (Pollock andClackson, T. et al. (1998) Proc. Natl. Acad. Sci. U.S.A.95:10437-10442). In one study, ligands that possess a synthetic “bump”in the FKBP-binding domain were shown to bind more tightly to a mutatedFKBP having a cavity formed by the removal of an aromatic side chain(i.e., harboring the substitution, F36V). Such engineered ligands bindpreferentially to the mutated FKBP12 compared to thewild-type/naturally-occurring protein by almost three orders ofmagnitude (Clackson, T. et al. (1998) Proc. Natl. Acad. Sci. U.S.A.95:10437-10442). Moreover, this family of ligands does not elicitundesired responses when administered to cultured cells or animals,including humans (Iuliucci, J. D. et al. (2001) J. Clin. Pharmacol.41:870-879).

FIG. 1A illustrates a method for conditionally controlling proteinstability using a fusion protein containing a destabilizing domain (DD)derived from FKBP fused in-frame to a protein of interest (POI). In theabsence of a stabilizing ligand, the destabilizing domain mediates thedegradation of the entire fusion protein. The addition of an appropriateligand stabilizes the destabilizing domain, greatly reducing degradationof the fusion protein. Using such FKBP polypeptides and ligands, asingle unstable ligand-binding domain is able to direct the degradationof a POI, avoiding the need to assemble the FKBP-rapamycin-FRAP/mTORternary complex to regulate protein function.

To identify FKBP variants (i.e., mutants) with a high affinity for thesynthetic FKBP ligand, SLF* (FIG. 1B) a cell-based screening assay wasused to screen a library based on the FKBP F36V gene sequence, which wascloned in-frame with yellow fluorescent protein (YFP) as a reporterprotein/protein of interest. In this manner, YFP fluorescence served asan indicator of FKBP stability. The FKBP-YFP fusion protein library(i.e., a library of N-terminal FKBP mutants) was introduced into NIH3T3fibroblasts using a retroviral expression system. Thetransfected/transduced cells were subjected to three rounds of sortingusing flow cytometry, as illustrated in FIG. 1C. The cells were treatedwith SLF* (FIG. 1B) in the first round of sorting. The fluorescent cellswere collected, cultured in the absence of ligand (second round), andthen cultured again in the presence of SLF* (third round), at which timeYFP-expressing cells were collected and the genomic DNA was isolated forsequence analysis (Example 7). All sequences analyzed maintained theF36V mutation (not reflected in the nomenclature), along with otherfrequently recurring amino acid mutations (Table 1).

Five variants (F15S, V24A, H25R, E60G, and L106P) were selected forfurther analysis. These variant FKBP-derived, ligand-responsivedestabilizing domains were separately transduced into NIH3T3 cells andassayed for stability in the presence and absence of a ligand calledShield1, which was a derivative of SLF* in which the carboxylic acid wasreplaced with a morpholine group at a position unlikely to interferewith FKBP binding (Example 8, FIG. 1B).

All five variants showed decreased levels of fluorescence with respectto a positive control, indicating that the variants obtained from thelibrary screen were destabilizing (FIG. 2A). The most destabilizingvariation, L106P, produced YFP fluorescence at a level of only 1-2%relative to the positive control. All FKBP-derived, ligand-responsivedestabilizing domain variants produced increased fluorescence signalwhen incubated in the presence of Shield1 (FIG. 2B). Variant V24A showedthe most efficient rescue (i.e., stabilization by Shield1) with an EC₅₀of about 5 nM. Variant L106P required a higher concentrations of Shield1to stabilize the YFP fusion protein (EC₅₀ 100 nM). YFP fluorescenceincreased at approximately the same rate in all the transfected cellsupon addition of Shield1, with maximum fluorescence being achieved at 24hours and stably maintained for at least an additional 48 hours withoutfurther addition of Shield1 (FIG. 2C).

Upon withdrawal of Shield1, distinct differences in fluorescence decayprofiles were observed among the FKBP-derived, destabilizing domainvariants (FIG. 2D), revealing a correlation between the rate ofdegradation and the degree of destabilization. Variant H25R, which isthe least destabilizing of this group, showed the slowest rate ofdegradation, whereas L106P, the most destabilizing of the five, wasdegraded most quickly, with protein levels becoming negligible withinfour hours. Similar results were obtained upon withdrawal of Shield1from C-terminal variant FKBP fusions, as shown in FIG. 12.

These results were confirmed by immunoblot analysis using antibodiesspecific for either FKBP (FIG. 2E) or YFP (data not shown), which wereincapable of detection in protein lysates from mock-treated cells, butdetectable in detected in protein lysates from Shield1-treated cells(Example 8). Incubation of the transfected cells with MG132 (FIG. 2F) orlactacystin (FIG. 9), which are drugs that inhibitubiquitin-proteasome-mediated protein degradation, inhibited degradationof the variant FKBP fusion proteins following the withdrawal of Shield1,indicating that degradation was mediated, at least in part, by theproteasome.

FIG. 3 shows the results of an experiment demonstrating the reversiblenature of small-molecule regulation of intracellular protein levels. Apopulation of NIH3T3 cells stably expressing the L106P-YFP fusionprotein was treated with different concentrations of Shield1 over thecourse of one week, and samples of the population were assayed forfluorescence by flow cytometry at the indicated time points. Predictedfluorescence is based on the dose-response experiment shown in FIG. 2B.FIG. 8 shows the kinetics of fluorescence in cells stably expressing oneof several FKBP-YFP fusion proteins upon addition of Shield1.

To compare the present methods with those known in art for regulatinggene expression, the rate of degradation of a protein of interestachieved using the FKBP-derived, destabilizing domain variants, wascompared to RNAi-mediated silencing of another endogenous gene, LaminA/C, a non-essential cytoskeletal protein commonly used as a control inRNAi experiments. Previous studies have shown more than 90% reduction inlamin A/C expression in HeLa cells 40 to 45 hours following transfectionof the cells with a cognate siRNA duplex (Elbashir, S. M. et al. (2001)Nature 411:494-498). In line with published results, HeLa cellstransfected with siRNA against lamin A/C showed a decrease in lamin A/Clevels after 24 hours, with a significant reduction in lamin A/Cobserved by 48 hours (FIGS. 2G and 10). In contrast, cells stablyexpressing L106P-YFP show nearly complete degradation of the fusionwithin 4 hours of removal of Shield1. These results demonstrate thatfusion of a destabilizing domain to a protein of interest dramaticallyreduces its stability in cultured cells, causing the protein of interestto be quickly degraded upon removal of the stabilizing ligand (Example8).

Further experiments demonstrated that NIH3T3 cells stably expressing theL106P-YFP variant produced YFP fluorescence in a dosage-dependent mannerwith respect to the amount of ligand present (Example 9). The resultsobtained using N-terminal destabilizing domains are shown in FIG. 2B.C-terminal destabilizing domains responded to Shield1 in adose-dependent manner comparable to N-terminal destabilizing domains,with EC₅₀ values ranging from 10 nM to 100 nM (Example 10, FIG. 11, andTable 2). Ligand-dependent protein stability was also observed inNIH3T3, HEK 293T, HeLa, and COS-1 cells that were transfected with theFKBP-derived fusion proteins (Example 11, Table 3), demonstrating thatligand-dependent stability is not restricted to one cell type.

FKBP variants were efficient in destabilizing proteins other than YFP,e.g., the kinases GSK-3β and CDK1, the cell cycle regulatory proteinssecurin and p21, and three small GTPases, Rac1, RhoA and Cdc42 (FIG.4A). All these N-terminal fusion proteins demonstrated Shield1-dependentstability, with the absence of Shield1 resulting in the degradation in aShield1-dependent manner (Example 12).

FKBP variants were also efficient in destabilizing proteins other thanYFP, e.g., the transcription factor CREB, or the small GTPases Arf6 orArf7, when the FKBP mutant is fused at the C-terminus of the protein ofinterest (FIG. 4A). All these C-terminal fusion proteins demonstratedShield1-dependent stability, with the absence of Shield1 resulting inthe degradation in a Shield 1-dependent manner (Example 12). As shown inFIG. 5, the destabilizing FKBP variants D100G (SEQ ID NO: 7) and L106P(SEQ ID NO: 6) also conferred Shield1-dependent stability to atransmembrane protein, CD8α, when fused at the C-terminus of thetransmembrane protein.

Moreover, cell morphology could be manipulated by the presence orabsence of Shield1 (Example 13). Shield1-treated cells displayed thepredicted morphologies, i.e., expression of RhoA induced the formationof stress fibers, expression of Cdc42 resulted in filopodia formation,and expression of Arl7 induced the shrunken cell phenotype (Heo, W. D.and Meyer, T. (2003) Cell 113:315-328), while mock-treatment withShield1 produced cells with fibroblast-like morphologies. TheseGTPase-dependent morphology changes were reversible, as treatment withShield1 followed by removal of Shield1 also produced cells withfibroblast-like morphologies. The penetrance of the observed phenotypewas high, with a large percentage of cells (>90%) exposed to a givenexperimental condition displaying the predicted behavior (data notshown).

The FKBP-derived destabilizing domains can be stabilized using Shield1as well as the commercially available ligand, FK506 (FIG. 13).

b. Drug-OFF System

The above-described FKBP-derived conditional protein stability systemuses a variant of FKBP to stabilize a POI in the presence of anappropriate ligand, and destabilize the POI in the absence of theligand, representing a “drug-ON” system. In a related embodiment of thesystem, a variant FKBP polypeptide sequence was used to destabilize aPOI in the presence of an appropriate ligand, and stabilize the POI inthe absence of the ligand, representing a “drug-OFF” system.

Variant FKBP polypeptide sequences having the desired properties wereidentified by preparing a library of sequences encoding FKBP (F36V) witha short, 20-amino acid diversity element (i.e., population of sequencesencoding different amino acids) fused at the C-terminus of FKBP. TheFKBP-diversity element sequences were fused to the C-terminus of YFP, asbefore, and the sequences encoding the YFP-FKBP-diversity element wereintroduced into NIH3T3 cells for screening. Five different clones wereidentified in which YFP could be detected by FACS analysis in theabsence of the ligand (Shield1) but was nearly undetectable in thepresence of the ligand (i.e., a 5 to 6-fold decrease in the presence ofligand; FIG. 6A). The sequence encoding the FKBP-diversity elementvariants were recovered from the cells and sequenced, revealing that allfive clones encoded the same diversity element sequence, namelyTRGVEEVAEGVVLLRRRGN (SEQ ID NO: 18). Thus, a FKBP polypeptide fused tothe selected diversity element functioned as a stabilizing domain in theabsence of ligand, rather than a destabilizing domain, as ischaracteristic of, e.g., the F36V/L106P FKBP mutant and other mutants.

The “drug-OFF” system offers some advantages over the “drug-ON” system.In particular, the “drug-OFF” system requires the presence of the ligandonly to destabilize the POI. Thus, where the POI is essential forviability, the absence of the ligand allows the protein to functionnormally. In contrast, in a “drug-ON” system, the ligand must be presentat all times, except where the effect of removing the POI is beingstudied. Since the cost of maintaining cells or animals in the presenceof ligand over a prolonged period of time can be considerable, the“drug-OFF” system can be more economical.

c. Use of the System in Other Eukaryotic Organisms

While the FKBP system was first characterized in mammalian cells,further experiments have demonstrated that the system works in eukaryoteparasites, particularly Toxoplasma and Plasmodium.

FIGS. 6B and 6C shows the results of experiments performed in aToxoplasma gondii system, in which an FKBP-derived stability-affectingdomain was fused to the N-terminus of YFP. Nucleotides expressing thefusion the fusion protein were introduced into T. gondii parasites,which were then used to infect HFF cells. The parasitophorous vacuolesin the infected cells were monitored by time lapse fluorescencemicroscopy following the addition of ligand (Shield1; FIG. 6B) orfollowing the removal of ligand (FIG. 6C). The indicated control linecorresponds to background fluorescence, while each of the other lines inthe graphs correspond to individual infected cells. The results showthat the levels of YFP increase in the presence of ligand and decreasein the absence of ligand, demonstrating that the conditional proteinstability system is effective in Toxoplasma.

In another illustration of use of the system in eukaryotic parasites,FIGS. 6D and 6E shows the results of an experiment performed in aPlasmodium falciparum. The immunoblot shows the levels of YFP fusionprotein in transfected cells, which are greatly increased in thepresence of ligand. In a related experiment, fusion proteins were madewith falcipain-2, a cysteine protease. Knocking out the falcipain-2 genecauses vacuole swelling resulting from the decreased ability of theorganisms to degrade hemoglobin. Fusion of falcipain-2 to the F36V/L106PFKBP mutant produced a conditional falcipain stabilization system. Morethan 5-times as many organisms demonstrated the swollen vacuolephenotype in the presence of ligand (Shield1), compared to the absenceof ligand.

d. Use of the System In Vivo to Control the Stability of Tumor Proteins

HCT116 cancer cells were transfected with nucleic acids encoding thereporter gene luciferase fused to an FKBP-derived stability-affectingprotein (F36V/L106P) mutant. Stable transformants were selected and usedto challenge SCID mice. SCID were challenged with tumor cells expressingluciferase fused to-derived FKBP F36V/L106P-derived stability-affectingprotein and treated with the indicated amounts of ligand (Shield 1), oruntreated (control), and bioluminescent signals were imaged over time asdescribed (Lin, A. H., et al., (2005) J. Immunol. 175:547-54; Luo, J.,et al. (2006) Proc. Nat'l. Acad. Sci. USA 103:18326-31; n=4-10 mice pertreatment group).

The graph in FIG. 7A shows that the amount of luminescence is dependenton ligand dose, and that luminescence peaks at about 10 hours followinga single injection of ligand. The graph shown in FIG. 7B shows thatrepeated administration of the ligand (10 mg/kg Shield1 every 48 hours)results in continued luciferase stabilization. These results demonstratethat the conditional protein stabilization systems work in vivo, wherethe ligand is delivered to cells via the blood stream of an animal.

In a related experiment, polynucleotide sequences encoding the FKBPL106P stability-affecting protein were fused to polynucleotidescorresponding to the N-terminus of a thermostable luciferase to obtainthe chimeric gene L106P-tsLuc, which was stably integrated into HCT116colon cancer cells. HCT116 cells expressing the resulting fusion proteinwere tested for conditional regulation of luciferase activity in vitrousing the ligand, Shield1 (not shown) and then implanted as xenograftsonto immunodeficient mice. Shield1 (or vehicle only as a control) wasdelivered intraperitoneally (ip) at a dose of 10 mg/kg and luciferaseactivity was measured by in vivo bioluminescence imaging as shown inFIG. 21A. Maximum expression levels were observed 12 hours followingtreatment with Shield1, with bioluminescent signals returned tobackground within 48 hours, suggesting that Shield1 is deliveredsystemically and is maintained at sufficient levels within target cellsto stabilize the fusion protein for a significant period of time beforebeing cleared. Quantification of the data shown in FIG. 21B demonstratedan approximate 10-fold increase in signal in the presence of Shield 1compared to the absence of Sheild1, demonstrating conditional control ofprotein stability via the stability-affecting protein. Mice bearingHCT116 L106P-tsLuc xenografts and treated with increasing doses ofShield1 (i.e., 3, 6, or 10 mg/kg) showed increasing bioluminescencelevels (FIG. 21C), demonstrating dose responsiveness.

Animals treated with Shield1 (10 mg/kg) periodically, i.e., once every48 hours, were also found to maintain increased bioluminescence levelsover the course of several weeks compared to animals not treated withShield1 (FIG. 21D), suggesting that a periodic low-dose treatmentregimen with Shield1 is sufficient to control protein stability in vivo.

Periodic administration of ligand is advantageous in terms of reducingreagent cost and reducing the handling of animals; however, theadministration of Shield1 did not appear to affect feeding behavior,body weight, or overall activity (not shown), indicating that the ligandis substantially non-toxic to animals even when delivered frequently.These observations are consistent with microarray analysis of mRNAlevels in cells treated with Shield 1, which demonstrated no appreciablecellular response to treatment.

e. Use of the System In Vivo to Control Tumor Burden

The experiments described above relate to the conditional stability of areporter protein in tumors cells. Further experiments demonstrated thatconditional stabilization of a protein could actually affect tumorburden.

Interleukin-2 (IL-2) is a cytokine that is instrumental in immuneresponse, inducing the differentiation and proliferation of a variety oflymphocyte populations (Gaffen, S. L. and Liu, K. D. (2004) Cytokine28:109-23). Recombinant IL-2 is approved for clinical treatment of renalcancers and is used in a variety of other cancer therapies.Polynucleotides encoding the L106P stability-affecting protein wereinserted between polynucleotides of the IL-2 gene that encode the signalpeptide and the remainder of the IL-2 protein to produce the chimericgene, L-L106P-IL-2, which was stably introduced into HCT116 cells. Thelevels of IL-2 secreted by the transfected HCT116 cells increased in adose-dependent manner depending on the concentration of Shield1 in themedia, with a dynamic range of approximately 25-fold (FIG. 22A).

Cells harboring the L-L106P-IL-2 chimeric gene, were implanted as axenograft onto mice (as above), and the ability of the cells toestablish tumors and proliferate in the presence and absence of ligand(Shield1) was monitored (FIG. 22B). Under these conditions, the HCT116L-L106P-IL-2 cells secreted stabilized IL-2, in a “drug-ON” manner.

Tumors in the untreated control animals continued to increase in sizeover the course of the experiment. Tumors failed to establish in micethat received HCT116 L-L106P-IL-2 cells and had been pretreated withShield1 and continued to be treated with Shield1 (10 mg/kg) every 48hours following implantation of the xenograft. Mice that were notpretreated with Shield1 prior to tumor implantation but were treatedwith Shield1 (i.p. at either 5 or 10 mg/kg every 48 hours) five daysfollowing tumor establishment showed tumor regression, with the size ofthe tumor eventually being reduced to that observed in the pretreatedanimals. By day 16, all Shield 1-treated groups displayed significantlyreduced tumor burden relative to untreated animals (p<0.05).

These results demonstrated that stabilization of IL-2 followingimplantation resulted in tumor regression, whereas stabilization of IL-2prior to and following implantation prevented tumor establishment.

Analysis of IL-2 levels within the tumors of treated and untreated miceconfirmed dose-dependent production of IL-2 at the tumor site in micetreated at 5 or 10 mg/kg Shield1 (FIG. 22C; p=0.032). In contrast,analysis of serum IL-2 levels showed significantly elevated levels inthe 10 mg/kg Shield1 group relative to tumor-bearing mice not receivingShield1 (p=0.0004), whereas IL-2 serum levels in mice treated with only5 mg/kg Shield1 were similar to those in control group (FIG. 22D). Theseobservations suggested that Shield1 addition produced a tunable,dose-dependent secretion of a cytokine in vivo, and that altering thedose of Shield1 can achieve different biological effects. A dose of 5mg/kg Shield1 was sufficient to produce the same robust anti-tumorresponse as 10 mg/kg Shield1, while avoiding the increase in IL-2 serumlevels associated with the higher ligand dose.

f. Viral Delivery of a Conditional Stabilization System to Control TumorBurden

A viral gene delivery system for the systemic treatment of cancer basedon a replication-selective (oncolytic) strain of vaccinia virus(hereafter vvDD) has been described (Thorne, S. H. et al., (2006)Science 311:1780-84). The deletion of the viral thymidine kinase genesand growth factor genes restrict the replication of this virus to cancercells (McCart, J. A. et al. (2001) Cancer Res. 61:8751-7). Intravenousdelivery of vvDD results in the initial infection of tumor and othercells, followed by rapid replication and spread of the virus in tumorcells and clearance in other cells. vvDD can deliver transgenes to tumorcells following systemic delivery, allowing the system to be used tostudy conditionally stabilized proteins.

Preliminary experiments established that infection of cultured cellswith vvDD did not affect the kinetics or dynamic range ofL106P-regulated protein stability, and that the ligand, Shield1, did notaffect the replication of the virus (data not shown). Strains of vvDDwere then constructed to express either L106P-tsLuc or L106P-reffluorescent protein (RFP) fusion proteins and ligand (Shield1)-dependentprotein activity (i.e., fluorescence) was measured in cultured cells(FIG. 23A). The results of ligand (Shield1)-dependent stabilization ofvvDD-delivered L106P-tsLuc in mice are shown in FIGS. 23B and 23C. Bestresults were obtained by allowing 72 hours for viral infection toestablish within the tumor cells and to be cleared from most other cellsin the mice before beginning Shield1 treatment to selectively stabilizethe protein of interest in the tumor cells.

To test the ability of a virally-delivered condition proteinstabilization system to regulate physiologically relevant or therapeuticproteins, vvDD was used to deliver a conditionally stable cytokine TNF-αprotein. While proven to exert cytotoxic effects against primary tumors,the systemic toxicity of TNF-α has limited its clinical use to localapplications (e.g., isolated limb or organ perfusion; Lucas, R. et al.(2005) Curr. Cancer Drug Targets 5:381-392). In addition, TNF-αpossesses antiviral effects to which the vaccinia strain Western Reserve(the basis for vvDD) is susceptible (Alcami, A. et al. (1999) J. Gen.Virol. 80:949-59).

An L-L106P-TNF-α chimeric gene was inserted into a strain of vvDDconstitutively expressing luciferase, and dose-dependent stabilizationof secreted TNF-α from cultured cells was verified (not shown). Micebearing large (150-250 mm³) subcutaneous HCT116 tumors were treated witha single intravenous injection of this virus or a PBS control. Ligand(Shield1; 10 mg/kg) was delivered i.p. to the mice every 48 hours,starting either 1 day prior to vvDD treatment or 3 days post-treatmentvvDD.

When Shield1 dosing began prior to virus delivery (causing allvirus-infected cells to secrete stable TNF-α, only modest levels ofvirus were observed in the tumor (FIG. 24A), as measured by viralluciferase expression. It is likely that secretion of TNF-α frominfected cells rapidly targets these cells for destruction, thuslimiting the ability of the virus to become establish within the tumorcells in which they normally proliferate. Despite the modest levels ofvirus observed in the tumors, the presence of ligand resulted insignificantly decreased tumor burden compared to animals treated withvirus alone (FIG. 24B, p<0.05), presumably due to the antitumor effectsof the ligand-stabilized TNF-α. Shield1 delivered alone had no effect ontumor burden.

When Shield1 administration was delayed until 3 days following virustreatment, the initial delivery and persistence of viral infection (asmeasured by luciferase expression within the tumor) were equivalent tothose observed in animals treated with virus alone (FIG. 4 a). In theseanimals, the antitumor effects observed following the administration ofligand were significantly greater than those observed in animalspretreated with ligand (p<0.05). Seven of the eight animals demonstratedcomplete and durable responses (FIG. 4 b), with no adverse physiologicaleffects observed, demonstrating the efficacy of a conditional proteinstability system to modulate tumor progression in vivo.

In a subsequent experiment, vvDD was used to deliver a conditionallystable interleukin-2 (IL-2) protein. An L-L106P-IL-2 chimeric gene wasinserted into a strain of vvDD constitutively expressing luciferase.Mice bearing large (150-250 mm³) subcutaneous HCT116 tumors were treatedwith a single intravenous injection of this virus or a PBS control.Ligand (Shield1; 10 mg/kg) was delivered intraperitoneally (i.p.) to themice every 48 hours, starting either 1 day prior to vvDD treatment or 3days post-treatment with vvDD (72 hours after administration of thevirus).

As was observed in studies with the L-L106P-TNF-α vvDD studies describedabove, and as shown in FIG. 25, constitutive IL-2 expression resulted inpoor viral infection within the tumor (at day 3) and more rapidclearance of the virus as compared to virus observed in mice which wereadministered Shield1 72 hours after administration of the vvDDL-L106P-IL-2 virus. This may be attributed to the innate immune responsebeing rapidly induced. There was increased initial delivery to the tumorover the first 72 hours when IL-2 is destabilized in the absence ofShield1 and delayed clearance of the virus from the tumor when IL-2 issubsequently upregulated or stabilized via administration of Shield1.

However, when measuring effects on mouse survival, constitutive IL-2expression was again more beneficial than no IL-2 expression. FIG. 26suggests that regulated IL-2 expression, in which no Shield1 wasadministered during the first 72 hours after injection of the vvDDL-L106P-IL-2 virus, allowed more virus to accumulate in the tumor andresulted in the greater percent survival of the treated animals.

It was also observed that by delaying clearance of the virus, and thusprolonging infection of the virus, an increase in Treg infiltration wasobserved (see FIG. 27).

g. Regulating IL-2 Expression to Enhance Cell-Based Immunotherapy.

NK-92 is a highly cytotoxic NK cell line which has been shown to exhibitsubstantial antitumor activity against a wide range of malignancies invitro as well as in xenografted SCID mice. This therapeutic activity isenhanced by systemic administration of IL-2. Many immune cell therapiesrequire administration of cytokines to enhance their benefits in vivo.Use of systemic delivery of recombinant cytokines is often toxic, and isexpensive. Studies were done to determine the effects of treatingtumor-bearing mice with NK-92 cells pre-infected with thevvDD-L-L106P-IL2 virus. As a control, mice were also treated with thevvDD control virus (not containing the L-L106P-IL2 fusion construct) andco-administered recombinant IL-2.

SCID mice bearing HCT116 tumors were treated with NK-92 cell therapy.Subcutaneous tumors were formed by injecting 5×10⁶ HeLa cellssubcutaneously into athymic nu-/nu- mice. Once formed (after 7 days),mice were treated with a single intravenous injection of (i) PBS; (ii)1×10⁷ NK-92 cells pre-infected (a 2-hour infection step) with 1×10⁷ PFUvaccinia with thymidine kinase deletion or (iii) 1×10⁷ NK-92 cellspre-infected (after 2 h infection step) with 1×10⁷ PFU vaccinia withthymidine kinase deletion and expressing L106P-IL2 (n=8 mice per group;NK-92 cells from ATCC). Group (ii) mice were treated every 48 hours withintraperitoneal injections of 1×10⁷ IU rhIL2 (international unitsrecombinant human IL-2), and group (iii) were treated every 48 h with 10mg/kg Shield 1. Tumor burden was followed by caliper measurement.

FIG. 28 shows injection of tumor-bearing mice with NK-92 cellspreinfected with vvDD-L-L106P-IL-2 followed by administration of Shield1equally as effective as NK-92 preinfected with control virus and furtheradministered IL-2. Furthermore, mice injected with NK-92 cellspreinfected with vvDD-L-L106P-IL-2 displayed fewer signs of toxicity.

4. Stability-Affecting Protein Derived from DHFR

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme involved in theregeneration of tetrahydrofolate from dihydrofolate, using NADPH. Thegoal was to use E. coli DHFR as a destabilizing domain (DD) when fusedat either the N or the C-terminus of a protein of interest (POI). SinceDHFR is an enzyme present in mammalian cells, mutants of DHFR havingreduced catalytic ability were used to minimize perturbations to theintracellular environment caused by the introduction of an exogenousDHFR. Two of these mutations, Y100I (SEQ ID NO: 14) and G121V (SEQ IDNO: 15), in addition to reducing enzymatic activity, also destabilizedDHFR relative to the wild-type sequence (SEQ ID NO: 13).

These DHFR mutants acted as destabilizing domains when fused to yellowfluorescent protein (YFP) at either the N- or the C-terminus of the YFPreporter (FIG. 14). While fusion of the mutated DHFR to either the N orC-terminus of the YFP reporter resulted in ligand-dependentstabilization of the YFP reporter in the presence of trimethoprim (TMP;indicated by “+”) compared to the absence of TMP (indicated by “−”), theeffect was most pronounced when the Y100I and G121V DHFR were fused tothe N-terminus of the YFP reporter (FIG. 15).

To further improve the ligand-dependent stabilization characteristics ofthe DHFR-derived destabilizing domains for use at the C-termini of POIs,error-prone PCR was used to generate a library of additional mutants ofDHFR, similar to manner in which mutants of FKBP were generated. TheYFP-DHFR fusion protein library (i.e., the library of C-terminal DHFRmutants) was introduced into NIH3T3 fibroblasts using a retroviralexpression system, as before, and the transduced cells were subjected tofour rounds of sorting using flow cytometry. The cells were cultured inthe absence of ligand in the first round of sorting. Low YFP-expressingcells were collected, cultured in the presence of 10 μM TMP for 24 hours(i.e., the second round of sorting), and then again cultured in thepresence of 1 μM TMP (i.e., the third round of sorting) to isolateYFP-expressing cells with increased ligand-dependent stabilization.

Cells that displayed fluorescence were cultured in the presence of TMP,washed free of TMP, and sorted about four hours later to isolate mutantswith fast kinetics of degradation in the fourth round of sorting. LowYFP-expressing cells (i.e., cell in which YFP was most degradedfollowing the removal of the TMP from the cell medium), were collected,and the genomic DNA was isolated for sequence analysis. In this manner,two additional DHFR-derived destabilizing domains, having increasedligand-dependent stabilization, were isolated from the library screen,i.e., one double-mutant (N18T/A19V; SEQ ID NO: 17) and one single-mutant(F103L; SEQ ID NO: 16).

When fused to the C-terminus of YFP, these mutants destabilized YFP inthe absence of TMP and stabilized YFP in the presence of TMP. As shownin FIGS. 16A and 16B, the N18T/A19V and F103L DHFR mutants wereeffectively stabilized by lower concentrations of TMP relative to theoriginal Y100I and G121V mutants. A comparison of the F103L, N18T/A19V,and Y100I DHFR mutants fused to the C-terminus of YFP is shown in FIG.17A. An immunoblot performed using an antibody specific for DHFRconfirmed that the amount of DHFR present in cells is increased in thepresence of ligand (+) compared to the absence of ligand (−) (FIG. 17B).

The kinetics of YFP-fusion protein decay following withdrawal of ligandis shown in FIG. 18A. The rate of decay of the C-terminal fusion proteinharboring the F103L DHFR mutant was more rapid that of the C-terminalfusion protein harboring the N18T/A19V DHFR mutant, although the levelsof both proteins were similar by eight hours following withdrawal of theligand. The kinetics of YFP-fusion protein stabilization following theaddition of ligand is shown in FIG. 18B. The amount of YFP detectable incells initially increased linearly following addition of ligand,eventually reaching a maximum level.

To further improve the ligand-dependent stabilization characteristics ofthe DHFR-derived, stability-affecting proteins for use at the N-terminiof POIs, error-prone PCR was used to generate a library of additionalmutants of DHFR, similar to manner in which mutants of FKBP weregenerated. Using the wild-type sequence as well as the Y100I and G121Vmutant sequences as the basis of the library, five double mutants wereidentified from the screen, namely H12Y/Y100I (SEQ ID NO: 19),H12L/Y100I (SEQ ID NO: 20), R98H/F103S (SEQ ID NO: 21) M42T/H114R (SEQID NO: 22), and 161F/T68S (SEQ ID NO: 23).

FIG. 19A shows the relative mean fluorescence intensity in cellsharboring these mutant N-terminal fusion proteins in the absence andpresence of ligand. While the Y100I mutant produced the greatest amountof stabilization, the difference between the levels of YFP in theabsence and presence of ligand were greater with the each of the mutantsH12Y/Y100I, H12L/Y100I, and R98H/F103S. FIG. 19B shows the results of adose response experiment, in which cells harboring the same mutants asused in the experiment shown in FIG. 19A were exposed to increasingamounts of ligand. Consistent with this result shown in FIG. 19A, fusionproteins harboring the Y100I mutant are more stable in the presence oflower concentrations of ligand (or no ligand) but are no more stable inthe presence of higher concentrations of ligand.

The kinetics of decay and stabilization of the N-terminal fusionproteins harboring the H12Y/Y100I, H12L/Y100I, and R98H/F103S mutants isshown in FIGS. 20A and 20B, respectively. The three fusion proteinsbehaved in a similar manner, although the maximum levels of theR98H/F103S fusion protein appeared to be higher than the others.

These results demonstrate that the DHFR-derived stability-affectingproteins can function as in the context of either an N-terminal fusionor a C-terminal fusion with a POI.

5. Exemplary Systems for Conditionally Stabilizing BiologicalMacromolecules

Ideal techniques for conditionally stabilizing biological macromoleculesare specific, fast, reversible, and tunable. Cell-permeable smallmolecules often deliver the latter three features but, apart from a fewwell-known exceptions, cell-permeable small molecules are typically notspecific for a single biological target. The ideal conditionalstabilization technology combines the specificity of reverse genetics(i.e., well-defined DNA changes in a large genomic background) with theconditionality of cell-permeable small molecules.

Using small libraries of FKBP and DHFR variants (20,000 to 30,000members) in combination with a convenient cell-based screening assay,several ligand stabilized destruction domains were identified, whichconferred ligand-dependent stability to a POI. A list of proteins thathave to date been stabilized using the methods and compositionsdescribed herein is provided in Example 12. The FKBP-deriveddestabilizing domains conferred ligand-dependent stability tocytoplasmic, nuclear, and a transmembrane protein, indicating that thepresent methods and compositions are generally applicable to the studyof protein function. Stability, and therefore function, of the fusionproteins was greatly increased upon addition of a cell-permeablehigh-affinity ligand. For example, when the most destabilizing FKBPvariants from the screen, i.e., FKBP L106P, was fused to YFP, the fusionprotein is expressed at only ˜1-2% of its maximum level in the absenceof the stabilizing ligand. This fusion protein is fully stabilized uponthe addition of 1 μM Shield1.

Variant and mutant FKBP proteins are exemplified by FKBP F36V (SEQ IDNO: 1) and the variants described in the text, Table 1 (N-terminalfusion proteins), and Table 2 (C-terminal fusion proteins). Exemplaryvariants have the substitutions F15S (SEQ ID NO: 2), V24A (SEQ ID NO:3), H25R (SEQ ID NO: 4), E60G (SEQ ID NO: 5), L106P (SEQ ID NO: 6),D100G (SEQ ID NO: 7), M66T (SEQ ID NO: 8), R71G (SEQ ID NO: 9), D100N(SEQ ID NO: 10), E102G (SEQ ID NO: 11), and K1051 (SEQ ID NO: 12). Astested, these variants included the F36V mutation (SEQ ID NO: 1);however, a similar mutation that accommodates a bulky side chain of acell-permeable ligand is expected to produce similar results. Moreover,the methods allow for the screening of additional mutations that yieldefficient single-ligand stabilized destruction domains.

A further mutant FKBP included additional amino acid sequence thataltered the behavior of the protein such that is stabilized a POI in theabsence of ligand and caused degradation of the POI in the presence ofligand. Thus the system can be used in a “drug-OFF” or “drug-ON”configuration. Such “drug-OFF” configurations may utilize a FKBP bindingdomain fused to a sequence substantially identical to that of SEQ IDNO:18.

The results obtained using DHFR variants suggests that Y100I (SEQ ID NO:14) G121V (SEQ ID NO: 15) variants, particularly with the N18T/A19V (SEQID NO: 17) or F103L (SEQ ID NO: 16), H12Y/Y100I (SEQ ID NO: 19),H12L/Y100I (SEQ ID NO: 20), and R98H/F103S (SEQ ID NO: 21)substitutions, are well-suited for use as single-ligand stabilizeddestruction domain. However, the methods allow for the screening ofadditional mutations, including those for operation in a “drug-ON”configuration, as well as in the exemplified “drug-OFF” configuration.

The abundance of variants obtained in the screens, as well as theability to use different ligand-binding domains, suggests that furtherrefinements in screening may lead to additional stability-affectingproteins selected for various properties (e.g., rate of degradation,potency of stabilization, subcellular localization, and the like).Moreover, the stability-affecting proteins work when fused to either theN- or the C-terminus of a POI, illustrating the modularity of thecomponents of the system.

The present systems work in different cell types, and work in cellculture and in animals. The system provides heretofore unprecedentedcontrol of the levels of preselected protein in cells, with excellentdose and temporal control. While the present methods have been describedwith reference to the FKBP and DHFR-derived destabilizing domains, otherdomains may be used. Preferred stability-affecting proteins modulate thedegradation of a fusion protein, as determined, for example, using thekinetic and immunological assays described herein. In some embodiments,the level or extent of destabilization of the destabilization domainfusion protein is dependent upon the amount of ligand administered tothe cell or to the animal expressing the destabilization domain fusionprotein.

Preferred stability-affecting proteins produce a 5, 10, 20, 30, 40, 50,60, or more-fold difference in the levels of a preselected protein thatcan be detected in cells or animals in the absence or presence ofligand. In some embodiments, the gene or allele encoding thenaturally-occurring POI (i.e., the native protein, not a fusion protein)is deleted or disrupted in the genome of the cells or animal in whichthe conditional protein stability system is used or replaced by a DNAencoding the fusion protein. In this manner, the only source of the POIis the conditionally stabilized fusion protein, allowing its function tobe studies in the absence of the interferingwild-type/naturally-occurring protein.

The conditional protein stability system can be used not only in vitro,but also in vivo to control the expression of a reporter gene ortherapeutic gene of interest. An exemplary therapeutic gene is IL-2 butthe present methods can be used control the expression of a wide varietyof genes involved in tumor suppression, metabolic regulation, cellsignaling, transcription, replication, apoptosis, and the like.

In some embodiments of the method, the strategy is “drug-ON,” in thatthe stabilizing ligand must be present for stabilization of the fusionprotein. However, if the POI exhibits a dominant negative phenotype, thesystem may be “drug-OFF,” in that addition of the ligand stabilizes thedominant negative fusion protein, which in turn inhibits the function ofits cellular target protein.

The stability-affecting proteins may encompass amino acid substitutionsthat do not substantially affect stability, including conservative andnon-conservative substitutions Preferably, the amino acid sequences ofthe peptide inhibitors encompassed in the invention have at least about60% identity, further at least about 70% identity, preferably at leastabout 75% or 80% identity, more preferably at least about 85% or 90%identity, and further preferably at least about 95% identity, to theamino acid sequences set forth herein. Percent identity may bedetermined, for example, by comparing sequence information using theadvanced BLAST computer program, including version 2.2.9, available fromthe National Institutes of Health. The BLAST program is based on thealignment method of Karlin and Altschul ((1990) Proc. Natl. Acad. Sci.U.S.A. 87:2264-68) and as discussed in Altschul et al. ((1990) J. Mol.Biol. 215:403-10; Karlin and Altschul (1993) Proc. Natl. Acad. Sci.U.S.A. 90:5873-77; and Altschul et al. (1997) Nucleic Acids Res.25:3389-3402).

Conservative amino acid substitutions may be made in the amino acidsequences described herein to obtain derivatives of the peptides thatmay advantageously be utilized in the present invention. Conservativeamino acid substitutions, as known in the art and as referred to herein,involve substituting amino acids in a protein with amino acids havingsimilar side chains in terms of, for example, structure, size and/orchemical properties. For example, the amino acids within each of thefollowing groups may be interchanged with other amino acids in the samegroup: amino acids having aliphatic side chains, including glycine,alanine, valine, leucine and isoleucine; amino acids havingnon-aromatic, hydroxyl-containing side chains, such as serine andthreonine; amino acids having acidic side chains, such as aspartic acidand glutamic acid; amino acids having amide side chains, includingglutamine and asparagine; basic amino acids, including lysine, arginineand histidine; amino acids having aromatic ring side chains, includingphenylalanine, tyrosine and tryptophan; and amino acids havingsulfur-containing side chains, including cysteine and methionine.Additionally, amino acids having acidic side chains, such as asparticacid and glutamic acid, can often be substituted with amino acids havingamide side chains, such as asparagine and glutamine.

The stability-affecting proteins may be fragments of the above-describeddestabilizing domains, including fragments containing variant amino acidsequences. Such fragments are readily identified using the assaysdescribed herein. Preferred fragments retain the ability to bind to astabilizing ligand with similar efficiency to the destabilizing domainsdescribed herein or with at least 90% efficiency, at least 80%efficiency, at least 70% efficiency, or even at least 50% efficiencywith respect to the described stability-affecting proteins.

Stabilizing ligands for use according to the methods described hereinare exemplified by SLF* (i.e.,1-[2-(3,4,5-trimethoxy-phenyl)-butyryl]-piperazine-2-carboxylic acid1-(3-carboxymethoxy-phenyl)-3-(3,4-dimethoxy-phenyl)-propyl ester) andShield1 (i.e.,1-[2-(3,4,5-trimethoxy-phenyl)-butyryl]-piperazine-2-carboxylic acid3-(3,4-dimethoxy-phenyl)-1-[3-(2-morpholin-4-yl-ethoxy)-phenyl]-propylester), both shown in FIG. 1B, and MaRap (C20-methallylrapamycin). Afeature of these FKBP ligands is that they contain a “bump” (i.e., abulky side-chain substituent) that prevents the ligand from binding towild-type (i.e., naturally-occurring) FRB domain of FRAP/mTor, therebyminimizing the biological effects associated with rapamycinadministering. The “bump” in the ligand corresponds to a “hole” (i.e., acompensatory, cavity-forming substitution or mutation) in the FRB domainof FRAP/mTor.

Other stabilizing ligands may be used according to the present methods.Such ligands include rapamycin-derived ligands containing other bulkyside-chains at positions of the molecule known to mediate binding toFKBP. As illustrated by the exemplary stabilizing ligands, theparticular side-chain is not critical, with both aliphatic and aromaticside-chains producing acceptable results. Numerous other bulky R-groupsare expected to give similar results, including but not limited toalkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic groups. The R-groups may contain a hydroxyl,alkoxy, substituted alkoxy, alkenoxy, substituted alkenoxy, cycloalkoxy,substituted cycloalkoxy, cycloalkenoxy, substituted cycloalkenoxy,aryloxy, substituted aryloxy, heteroaryloxy, substituted heteroaryloxy,heterocyclyloxy, substituted heterocyclyloxy and secondary or tertiaryamine, (i.e., —NR′R″ where each R′ or R″ is independently selected fromhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, substituted heterocyclic, etc.

A related aspect of the methods and compositions are cells transfectedwith nucleic acids encoding a fusion protein comprising a protein ofinterest fused in frame to a stability-affecting protein. Expression ofthe fusion protein may be driven by the endogenous promoter, ideallyreproducing the spatial and temporal expression patterns of theunmodified gene. The cells may be transfected, e.g., using an expressionvector, or transduced (i.e., infected) using a viral vector, includingbut not limited to a vector derived from a retrovirus (e.g., alentivirus), herpesvirus, pox virus, adenovirus, adenoassociated virus,or an RNA virus, such as poliovirus, flavivirus, alphavirus, or thelike. The exemplary viral vector was based on a retrovirus.

The system was shown to be effective eukaryotic cells, includingmammalian cells and protozoan parasites; therefore, the system can beexpected to work in various eukaryotic cells, including those of humans,primates, rodents, dogs, cats, horses, cows, sheep, insects, amphibians,and apicomplexan parasites. The cells may be in culture or in a livingorganism. As noted above, the wild-type or naturally-occurring gene orallele encoding the POI may be deleted to facilitate study of theconditionally stabilized POI.

The present methods and compositions also allow the creation oftransgenic animals harboring engineered alleles that direct theexpression of a ligand-stabilized POI. Expression of the fusion proteinmay be driven by the endogenous promoter, ideally reproducing thespatial and temporal expression patterns of the unmodified gene. Theligand may be administered regularly from an early age (including inutero) to stabilize the fusion protein until the mice achieve aspecified age, at which time withdrawal of the ligand results in a therapid degradation of the fusion protein. Unlike Cre-mediated genedisruption (see Background section), this method is reversible, simplyby reinitiating the administration of the ligand, allowing the rapid,reversible, and conditional control of protein function in a complexsystem.

The ability to specifically and conditionally stabilize a POI in a cellwill enable the study of many proteins to determine their biologicalfunction and importance in a cell. The present methods and compositionrepresent a significant improvement over current methods of conditionalprotein regulation.

6. Kits of Parts

The methods and compositions described herein may be packaged togetherwith instructions for use, as in a kit of parts. Preferred kits of partsinclude nucleic acids encoding stability-affecting proteins, one or moreligands, and instructions for use. The instructions may containinformation relating the inserting (i.e., “cloning”) a POI into aplasmid, in-frame with a stability-affecting protein. The instructionsmay also include dosing recommendations and hardware, such as syringes,to deliver the fusion protein to an organism or to cells.

EXAMPLES

The following examples are illustrative in nature and are in no wayintended to be limiting. Examples relating to generating and screeningstability-affecting proteins apply generally to FKBP, DHFR, and otherpolypeptides that can be used as stability-affecting proteins.

Example 1 FKBP Library Generation

Diversity in the FKBP sequence was generated using a combination oferror-prone PCR and nucleotide analog mutagenesis. Primers for mutagenicPCR were designed to anneal upstream of the 5′ restriction site to beused for cloning the mutagenesis products into the pBMN iHcRed-tandemretroviral expression vector and downstream of the 3′ restriction site.Three independent condition sets were used to generate diversity.Condition set “A” utilized 4 ng template, 0.5 μM of each oligonucleotideprimer, 5 units Taq polymerase, 5 mM MgCl₂, 0.2 mM MnCl₂, 0.4 mM dNTPsin equal ratio and an excess of 0.2 mM dATP and dCTP. Condition set “B”was identical to A except that dGTP and dTTP were present in excess.Condition set C utilized the non-natural nucleotides 8-oxo-dGTP and dPTPto encourage nucleotide misincorporation (Zaccolo, M. et al. (1996) J.Mol. Biol. 255:589-603). The FKBP libraries were pooled and ligated intothe pBMN iHcRed-t retroviral expression vector, affording a librarycontaining ˜3×10⁴ members.

Example 2 FKBP Synthetic Ligands

SLF* and Shield1 were synthesized essentially as described (Holt et al.,1993; Yang et al., 2000).

Example 3 Cell Culture, Transfections, and Transductions

The NIH3T3 cell line was cultured in DMEM supplemented with 10%heat-inactivated donor bovine serum (Invitrogen), 2 mM glutamine, 100U/ml penicillin and 100 μg/ml streptomycin. All other cell lines werecultured with 10% heat-inactivated fetal bovine serum (Invitrogen), 2 mMglutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.

The ΦNX ecotropic packaging cell line was transfected using standardLipofectamine 2000 protocols. Viral supernatants were harvested 48 hrspost-transfection, filtered and concentrated 10-fold using an AmiconUltra centrifugal filter device (Millipore, 100-kDa cut-off). NIH3T3cells were incubated with the concentrated retroviral supernatantssupplemented with 4 μg/ml polybrene for 4 hrs at 37° C. Cells werewashed once with PBS and cultured in growth media for 24-36 hrs to allowfor viral integration, then assayed as described.

HeLa cells were plated at 7×10⁴ cells per well of a 24-well plate 12hours prior to transfection. Cells were transfected with either 200 ngSilencer® Lamin A/C siRNA (Ambion) or a negative control siRNA using theGeneSilencer protocol. Cell lysates were immunoblotted with ananti-lamin A/C antibody (Clone 14, BD Transduction Laboratories).

Example 4 Flow Cytometry

Twenty-four hours prior to analysis, transduced NIH3T3 cells were platedat 1×10⁵ cells per well of a 12-well plate and treated as described.Cells were removed from the plate using PBS+2 mM EDTA, washed once withPBS, and resuspended in 200 μL PBS. Cells were analyzed at the StanfordShared FACS Facility using FlasherII with 10,000 events represented.

Example 5 Protein-of-Interest Origin and Antibodies

Proteins tested as fusions to destabilizing domains were of thefollowing origin, and the following antibodies were used forimmunoblotting: Arf6 Q67L (human, 3A-1, Santa Cruz Biotechnology); Arl7Q72L (human, BC001051, Protein Tech Group, Inc.); Cdc42 Q61L (human, P1,Santa Cruz Biotechnology); CD8α(mouse, 5H10, Caltag Laboratories); CDK1(human, H-297, Santa Cruz Biotechnology); CREB (mouse, 86B10, CellSignaling Technology); FKBP (human, 2C1-97, BD PharMingen);GSK-3β(mouse, 0011-A, Santa Cruz Biotechnology); Hsp90 (mouse, 68, BDTransduction Laboratories); p21 (human, H-164, Santa CruzBiotechnology); Rac1 Q61L (human, C-11, Santa Cruz Biotechnology); RhoAQ63L (human, 26C4, Santa Cruz Biotechnology); Securin (human, Z23.YU,Zymed Laboratories); YFP, Aequorea Victoria (JL-8, Clontech).

Example 6 Phalloidin Staining and Microscopy

NIH3T3 cells stably expressing constitutively active GTPases fused todestabilizing domains were treated with 1 μM Shield1 for 24 hr. At thistime, cells were washed once with PBS, plated at 8×10³ cells in 4-wellLabTek Chambered coverglass (NUNC) coated with 1 mg/ml poly-D-lysine(Sigma), along with mock-treated transduced cells and transduced cellstreated with 1 μM Shield1. Cells were cultured for 24 hr in 10% DBS,then cultured in serum-free media for 12 hr. Cells were then washed withPBS, fixed in 4% paraformaldehyde for 15 min, permeabilized in 0.2%Triton X-100 for 5 min, stained with 1 μg/ml Alexa Fluor 488-conjugatedphalloidin (Invitrogen; A12379) in PBS for 20 min, and washed with PBS.Fixed cells were imaged using a Bio-Rad Radiance 2100 confocalmicroscope.

Example 7 Identification of Ligand-Responsive Destabilizing Domain

To identify FKBP variants (i.e., mutants) with a high affinity for thesynthetic FKBP ligand SLF* (FIG. 1B) a cell-based screening assay wasused in which a library based on the FKBP F36V gene sequence wasgenerated using error-prone PCR, and then cloned in-frame in front ofyellow fluorescent protein (YFP). Measurement of the fluorescence of YFPserved as an indicator of FKBP stability.

A Moloney murine leukemia retroviral expression system was used tostably integrate this library of DNAs encoding FKBP-YFP fusion proteinsinto NIH3T3 fibroblasts. The transduced cells were subjected to threerounds of sorting using flow cytometry. In the first round, cells weretreated with the FKBP ligand SLF* (5 μM, FIG. 1B) for 24 hours prior tosorting. The fluorescent cells were collected and further cultured inthe absence of ligand for 60 hours. Reanalysis revealed thatapproximately 5% of the cell population exhibited decreased fluorescencelevels, indicating that the majority of the sequences were eitherunmutated or contained mutations that did not affect stability of thefusion protein. This small population of cells exhibiting decreasedfluorescence was collected and cultured again in the presence of SLF* (5μM) for 24 hours, at which time YFP-expressing cells were collected andthe genomic DNA was isolated. The sequence analysis of 72 FKBP-derivedlibrary clones (Table 1) revealed several frequently recurring mutationsthat were distributed fairly evenly over the primary amino acidsequence. All sequences maintained the F36V mutation. To validate thescreening method and to further characterize the FKBP-derivedligand-responsive destabilizing domains, we chose five variants (F15S,V24A, H25R, E60G, and L106P) for further analysis.

TABLE 1 Shield 1-dependent N-terminal FKBP mutants isolated from libraryscreen. Clone No. Mutations Clone No. Mutations Identity of mutations1-37 0 none 38 0 stop codon introduced 39 0 dropped base 40 0 mixedsequence 41 1 F15S 42 1 F15S 43 1 V24A 44 1 K34R 45 1 S38P 46 1 F46L 471 V63F 48 1 M66V 49 1 R71S 50 1 P78T 51 1 D79G 52 1 A81V 53 1 E102G 54 1L106P 55 2 F15S, N43S 56 2 Y26H, Q53R 57 2 G28R, E31G 58 2 F48I, E60G 592 G51D, S77P 60 2 E54G, F99L 61 2 Q65R, L106P 62 3 V2A, L50A, L106A 63 3T6A, V24A, I91A 64 3 Q3R, N43S, G69S 65 3 K44E, E60G, V63A 66 3 W59R,E60G, I76M 67 4 R13H, V24A, K35A, M49A 68 6 S8P, G28R, L30P, S39P, F99L,D100G 69 7 F15S, H25R, K47G, K73R, I76V, D79G, I90V 70 10 H25R, M29T,L30P, D32G, P45S, F48L, K52E, S67G, L104P, L106P 71 11 H25R, M29T, L30P,D32G, P45S, F48L, K52E, E54G, S67G, L104P, L106P 72 16 I7T, S8P, P9L,D11N, T14A, F15L, H25Y, L30P, D37N, D41N, A64T, I76M, G83D, T85A, I91V,F99V

Example 8 Characterization of Ligand-Responsive Destabilizing Domain

The variant FKBP-derived, ligand-responsive destabilizing domains wereassayed for stability in the presence and absence of a derivative ofSLF* in which the carboxylic acid is replaced with a morpholine group(FIG. 1B). This functional group is commonly appended to drug-likemolecules to improve their pharmacokinetic properties, and was added toSLF* at a position unlikely to interfere with FKBP binding. The modifiedSLF*-derived, cell-permeable FKBP ligand was designed to protect anotherwise unstable protein domain from degradation, and was thereforecalled Shield1 (Shield 1).

Each variant FKBP-derived, ligand-responsive destabilizing domain wasseparately transduced into NIH3T3 cells, and YFP fluorescence levelswere measured in the absence of Shield1 (FIG. 2A). All five mutantsshowed decreased fluorescence levels with respect to a positive control,indicating that the variants obtained from the library screen weredestabilizing. The most destabilizing variation, L106P, produced YFPfluorescence at a level of only 1-2% relative to the positive control.All FKBP-derived, ligand-responsive destabilizing domain variantsproduced increased fluorescence signal when incubated in the presence ofShield1 (FIG. 2A). The difference in the efficiency of rescue (i.e.,stabilization by Shield1) varied by over an order of magnitude, as shownin FIG. 2B. Variant V24A showed the most efficient rescue, with theextracellular concentration of Shield1 required to obtain 50% of themaximum YFP signal being 5 nM (i.e., EC₅₀˜5 nM). The more destabilizingL106P variant required higher concentrations of Shield1 (EC_(50˜100) nM)to stabilize the YFP fusion protein.

In a kinetic study of NIH3T3 cells stably expressing each of the fiveFKBP-derived, ligand-responsive destabilizing domain variants, YFPfluorescence increased at approximately the same rate upon addition ofShield1, with maximum fluorescence achieved at 24 hours and stablymaintained for at least an additional 48 hours without further additionof Shield1 (FIG. 2C). These results suggest that, upon addition ofShield1, these FKBP mutants are able to adopt a conformation thatapproximates the stability of the wild type protein, and that increasesin fluorescence are mainly a function of the rate of protein synthesisand/or YFP maturation within the cell. In a related experiment, NIH3T3cells transduced with the FKBP L106P-YFP fusion (hereafter L106P-YFP)were treated with various concentrations of Shield1 and YFP fluorescencewas monitored as a function of time (FIG. 2C). YFP expression isobserved within 15 min, and cells treated with lower concentrations ofShield1 reach steady state expression levels more rapidly than cellstreated with higher concentrations of Shield1.

Upon withdrawal of Shield1, distinct differences in fluorescence decayprofiles were observed among the FKBP-derived, ligand-responsivedestabilizing domain variants (FIG. 2D), revealing a correlation betweenthe rate of degradation and the degree of destabilization. Variant H25R,which is the least destabilizing of this group, showed the slowest rateof degradation, whereas L106P, the most destabilizing of the five, wasdegraded most quickly, with protein levels becoming negligible withinfour hours.

To correlate YFP fluorescence with intracellular protein levels and tolook for evidence of partial proteolysis, cells stably expressing eachdestabilizing domain fused to YFP were either mock-treated or treatedwith Shield1. Cell lysates were prepared and used for immunoblotanalysis along with antibodies specific for either FKBP (FIG. 2E) or YFP(data not shown). Neither antibody was capable of detecting protein inlysates from mock-treated cells, whereas the fusion protein was detectedin Shield1-treated cells. Cells transformed with either the F15S orL106P variant were also examined using fluorescence microscopy, whichdemonstrated Shield1-dependent fluorescence (data not shown).

The mechanism of degradation was examined for the F15S and L106Pvariants. Since the ubiquitin-proteasome system is a major mediator ofintracellular protein degradation (Pickart, C. M. (2004) Cell116:181-190; Bence, N. F. et al. (2001); Hicke, L. and Dunn, R.(2003)Annu. Rev. Cell Dev. Biol. 19:141-172). Science 292:1552-1555;),the cells expressing either the FKBP-derived, ligand-responsivedestabilizing domain variants F15S or L106P were incubated with MG132(FIG. 2F) or lactacystin (FIG. 9), which inhibitubiquitin-proteasome-mediated protein degradation. The inability of thecells to degrade the variant FKBP fusion proteins following thewithdrawal of Shield1, indicating that degradation was mediated, atleast in part, by the proteasome.

RNAi has become a widely used tool for reducing intracellular levels ofa protein of interest. The rate of RNAi-mediated silencing of anendogenous gene was compared to the rate of degradation achieved throughthe fusion of a protein of interest to the above-described destabilizingdomain. Lamin A/C is a non-essential cytoskeletal protein commonly usedas a control in RNAi experiments. Previous studies have shown more than90% reduction in lamin A/C expression in HeLa cells assayed 40 to 45hours after transfection of the cells with a cognate siRNA duplex(Elbashir, S. M. et al. (2001) Nature 411:494-498), which suggests thatthe half-life of the lamin A/C proteins is about 10-12 hours. Thishalf-life is significantly shorter than that of YFB, which is 26 hrs(Corish, P. and Tyler-Smith, C. (1999) Protein Eng. 12:1035-1040 andTyler-Smith, 1999). HeLa cells transfected with siRNA against lamin A/Cshowed a decrease in protein levels after 24 hours, with a significantreduction in lamin A/C observed by 48 hours (FIG. 2G, FIG. 10). Incontrast, cells stably expressing L106P-YFP show nearly completedegradation of the fusion within 4 hours of removal of Shield1. Theseresults demonstrate that fusion of a destabilizing domain to a proteinof interest dramatically reduces its stability in cultured cells,causing the protein of interest to be quickly degraded upon removal ofthe stabilizing ligand.

Example 9 Dose-Dependent Regulation of Intracellular Protein Levels

To determine the ability of the variant FKBP fusion proteins, incombination with Shield1, to modulate the levels of YFP in adose-dependent manner, NIH3T3 cells stably expressing the L106P-YFPvariant were exposed to different concentrations of Shield1 over thecourse of one week (FIG. 3). The smooth line (i.e., having no datapoints indicated by squares) are the predicted YFP levels based on thedose-response curve shown in FIG. 2B, as measured by flow cytometry.

Example 10 Identification and Characterization of C-TerminalDestabilizing Domains

A screen of a YFP-FKBP library (reversed compared to the previousorientation of FKBP and YFP) was performed to identify candidateC-terminal destabilizing domains (Table 2). Six FKBP variants (M66T,R71G, D100G, D100N, E102G, and K105I) were selected for furtheranalysis. Overall, destabilizing domains fused to the C-terminus of YFPare less destabilizing than their N-terminal counterparts (Table 1). Forexample, when the L106P mutant is fused to the N-terminus of YFP(L106P-YFP), fluorescence is only ˜1-2% of that observed in the presenceof Shield1. However, when the orientation is reversed (YFP-L106P),fluorescence in the absence of Shield1 is ˜10% of that observed in itspresence.

TABLE 2 Shield1-dependent C-terminal FKBP mutants isolated from libraryscreen. Clone No. mutations Identification of mutations 1-5 0 None  6 0Stop codon introduced 7-8 0 Incomplete sequence  8-12 0 Mixed sequence13 1 G1R 14 1 L30P 15 1 M66T 16 1 D100G 17 1 D100N 18 1 E102G 19 1 E102G20 1 E107G 21 2 E5K, R71G 22 2 E5K, R71G 23 2 D11G, K73R 24 2 Q20L, T27A25 2 T21A, H25R 26 2 C22F, M29T 27 2 M29T, D100G 28 2 E31G, E107G 29 2K34Q, Q70R 30 2 S67G, Q70R 31 2 G89S, K105I 32 3 V4M, G33R, G58S 33 3D11A, D32G, K44R 34 3 D11G, N43D, D79G 35 3 D11G, R13C, F48L 36 3 G19D,K35R, K105E 37 3 E31G, R71G, K105E 38 3 K35R, G69S, I76V 39 3 E61G,H94R, K105R 40 3 D79G, P93S, D100R 41 3 D79G, P93S, D100R 42 4 T6A, I7T,T14I, M66V 43 4 T21A, N43D, A72V, E107G 44 4 M29T, E31K, K52R, T75A 45 4R42G, K52R, D79G, E107G 46 5 I7T, M29V, F48L, T85A, K105R 47 7 Q3R,F15S, T21A, K44E, K73E, P88T, K105R 48 8 T6S, P9S, M29V, K34R, R42G,Q53R, K73R, D79G

Nonetheless, C-terminal destabilizing domains respond to Shield1 in adose-dependent manner comparable to N-terminal destabilizing domains,with EC₅₀ values ranging from 10 nM to 100 nM (FIG. 11). As observedwith N-terminal destabilizing domains, all variants exhibit nearlyidentical rates of increase in fluorescence upon addition of Shield1,regardless of the degree of instability conferred (not shown).

Example 11 Ligand-Dependent Stability in Multiple Cells Lines

Destabilizing domains fused to either the N- or C-terminus of YFP werealso transfected into several different cells lines, i.e., NIH3T3, HEK293T, HeLa, and COS-1 cells, to assess the behavior of the FKBP-derived,ligand-responsive destabilizing domain variants in different cells.Shield1-dependent fluorescence was observed in all cell lines (Table 3),demonstrating that ligand-dependent stability is not restricted to onecell type. The FKBP-derived destabilizing domains can be stabilizedusing Shield1 as well as the commercially available ligand, FK506 (FIG.13). However, unlike Shield 1, FK506 perturbs the cellular environmentby inhibiting calcineurin.

TABLE 3 Fluorescence of FKBP-YFP fusions (N-terminal or C-terminal) intransiently transfected cell lines in the absence of Shield1. % ResidualYFP Fluorescence* FKBP-YFP YFP-FKBP F15S L106P D100G L106P NIH3T3 7 8 1616 HEK 293T 7 5 15 19 HeLa 8 6 9 12 COS-1 12 19 22 26 *Data arepresented as the average mean fluorescence intensity relative to that ofthe maximum fluorescence intensity observed for the individual mutant.The experiment was performed in duplicate.

Example 12 Ligand-Dependent Stability for a Variety of Proteins

To show that FKBP variants are efficient in destabilizing proteins otherthan YFP, the F15S and L106P variants were fused at the N-termini, tothe kinases GSK-3β and CDK1, the cell cycle regulatory proteins securinand p21, and three small GTPases, Rac1, RhoA and Cdc42 (FIG. 4A). Allthe fusion proteins demonstrated Shield1-dependent stability, as was thecase for YFP. The absence of Shield1 resulted in the degradation of CDK1(an otherwise stable protein) as well as p21 and securing (cell cycleregulators with relatively short half-lives; Nigg, E. A. (2001) NatureRev. Mol. Cell. Biol. 2:21-32). Shield1-dependent stability of fusionproteins containing the D100G or

L106P destabilizing domain variants fused to the C-terminus of thetranscription factor CREB, or the small GTPases, Arf6 and Arl7, (FIG.4B), was also observed. To date, about 20 fusion proteins have beentested and all demonstrate ligand-dependent stability (Table 4). Anadditional example is CD8α, a transmembrane glycoprotein found on thesurface of T cells, which was able to be detected on the surface ofcells by flow cytometry (FIG. 5), in a Shield 1-dependent manner. Asshown in FIG. 5, the destabilizing FKBP variants D100G and L106P alsoconferred Shield1-dependent stability to a transmembrane protein, CD8α,when fused at the C-terminus of the transmembrane protein. Here, NIH3T3cells stably expressing the fusion proteins were divided into threepools (groups). The first group (−) was mock-treated, the second group(+) was treated with 1 μM Shield1 for 24 hrs, and the third group (+/−)was treated with 1 μM Shield1 for 24 hrs, and then washed with media andcultured for 24 hr in the absence of Shield1. Live cells were thenprobed with a FITC-conjugated anti-CD8α antibody and assayed by flowcytometry. Data are presented as the average mean fluorescence intensity±SEM from an experiment performed in triplicate.

TABLE 4 Proteins destabilized by FKBP Destruction Domains 1 yellowfluorescent protein (YFP) 2 glycogen synthase kinase-3β 3 securin 4p21^(WAF/CIP) 5 Rac1 6 Cdc42 7 RhoA 8 cAMP response element bindingtranscription factor (CREB) 9 cyclin-dependent kinase 1 (CDK1) 10 Arf611 Arl7 12 cyclin B1 13 firefly luciferase 14 Oct3/4 15 Sox2 16 Nanog 17c-Myc 18 Klf4 19 Aid 20 Apobec1 21 interleukin-2

Example 13 Ligand-Dependent Control of Cellular Phenotypes

Expression of constitutively active small GTPases causeswell-characterized changes in cellular morphology (Heo, W. D. and Meyer,T. (2003) Cell 113:315-328). To determine if FKBP-derived destabilizingdomains, in combination with Shield1, could affect cell morphology bymodulating GTPases levels, several small GTPases (i.e., RhoA, Cdc42, orArl7) were fused to the destabilizing domains (FIGS. 4A and 4B). NIH3T3cells were individually transduced with the L106P-RhoA, L106P-Cdc42, orArl7-L106P (note arrangement of fusions), and then mock-treated ortreated with Shield1, and visualized using confocal microscopy (notshown). Shield1-treated cells displayed the predicted morphologies,i.e., expression of RhoA induced the formation of stress fibers,expression of Cdc42 resulted in filopodia formation, and expression ofArl7 induced the shrunken cell phenotype (Heo, W. D. and Meyer, T.(2003) Cell 113:315-328). Mock treatment with Shield1 produced cellswith fibroblast-like morphologies. These GTPase-dependent morphologychanges were reversible, as treatment with Shield1 followed by removalof Shield1 also produced cells with fibroblast-like morphologies. Thepenetrance of the observed phenotype was high, with a large percentageof cells (>90%) exposed to a given experimental condition displaying thepredicted behavior (not shown).

Example 14 Cloning and Transfection of Luciferase and IL-2 Genes

Thermostable luciferase or the human IL-2 gene were cloned into pBMNL106P iBlasticidin and used to generate amphotropic retrovirus(Banaszynski, L. A. et al. (2006) Cell. 126:995-1004). HCT116 cells wereincubated with retrovirus and polybrene (6 μg/mL) for 4 hrs at 37° C.and then selected with Blasticidin (5 μg/mL). Cells grown in 96-wellplates (2×10⁴ cells/well) were treated with Shield1 as indicated andeither bioluminescence measured using an IVIS 50 (Xenogen Product fromCaliper Life Sciences) following luciferin addition (300 μg/mL), ormedia collected for ELISA.

Example 15 Mouse Models

SCID or CD1 nu-/nu- mice (Charles River Co.) received subcutaneousdorsal injections of ˜1×10⁷ cells, and tumors were allowed to establishas indicated. Animals were given an intraperitoneal injection ofluciferin (225 mg/kg), anesthetized (2% isoflurane), and placed on thewarmed stage (37° C.) of an IVIS 100 or IVIS 200 (Xenogen Product fromCaliper Life Sciences) for imaging. Light produced was measured asphotons/sec for designated regions of interest as described. Tumorvolumes were also determined by caliper measurement, and mice weresacrificed when tumors reached 1.44 cm³ for survival assays. In someexperiments, serum samples were collected by retino-orbital bleedingsand tumors collected post-mortem and homogenized for ELISA assay ofcytokines. All experiments were run with institutional IACUC approval.

Example 16 Vaccinia Virus Strains

CV1 cells were transfected with pSC-65 p7.5 L106P-tsLuc or pSC-65 p7.5L106P-TNF-α pSE/L Luc and simultaneously infected with viral growthfactor deleted Western Reserve Vaccinia (VSC20). Cassettes wereintegrated into the viral thymidine kinase gene by homologousrecombination and selected by resistance to bromodeoxyuridine on 143BTK⁻ cells. Single viral plaques were purified in 143B TK⁻ cells. Thesame method was used to generate vaccinia virus having the L-L106P-IL-2construct.

Example 17 vvDD Assays in Cultured Cells

HCT116 cells in a 96-well plate (2×10⁴ cells/well) were incubated withvvDD carrying an L106P fusion (MOI>1) for 1 hr at 37° C. Virus wasremoved and cells were treated with Shield1. For luminescence assays,cells were incubated with luciferin (300 μg/mL) and imaged using an IVIS50 (Xenogen product from Caliper Life Sciences). TNF-α in cell culturemedia was detected by ELISA.

Example 18 Statistical Analyses

Two-tailed, unpaired Student's T-tests were used, except for comparisonof survival curves, when Gehan-Breslow-Wilcoxon test was used. Resultswere considered significant when p<0.05.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

1. An in vivo conditional protein stability system comprising: a nucleicacid sequence encoding a fusion protein that comprises a protein ofinterest fused to a single-polypeptide chain, ligand-dependent,stability-affecting protein derived from a naturally-occurring ligandbinding protein, and a ligand that binds to the stability-affectingprotein to modulate stability of the stability-affecting protein whereinupon introduction of the nucleic acid sequence to a eukaryotic cell thefusion protein is expressed and the stability of the fusion protein canbe modulated by administering the ligand to the eukaryotic cell.
 2. Thesystem of claim 1, wherein the single-polypeptide chain,ligand-dependent, stability-affecting protein is a FKBP variant proteinand the naturally-occurring ligand binding protein is a wild-type FKBPprotein.
 3. The system of claim 1, wherein the protein of interest isTNF-α or IL-2.
 4. The system of claim 1, wherein the eukaryotic cellsare stably transformed with the nucleic acid.
 5. The system of claim 4,wherein the stably transformed eukaryotic cells are implanted in ananimal.
 6. The system of claim 1, wherein the nucleic acid sequence isin a viral vector.
 7. The system of claim 6, wherein the viral vector isa vaccinia virus.
 8. The system of claim 6, wherein thesingle-polypeptide chain, ligand-dependent, stability-affecting proteinis a FKBP variant protein and the naturally-occurring ligand bindingprotein is a wild-type FKBP protein.
 9. The system of claim 6, whereinthe protein of interest is TNF-α or IL-2.
 10. The system of claim 6,wherein the administering the ligand to the cells is by injecting theligand into the animal intraperitoneally or intravenously.
 10. A methodfor modulating stability of a protein of interest in vivo comprisingintroducing into a eukaryotic cell a nucleic acid comprising apolynucleotide which encodes a fusion protein wherein the fusion proteincomprises a protein of interest and a single-polypeptide chain,ligand-dependent, stability-affecting protein derived from anaturally-occurring ligand binding protein, and administering a ligandto the eukaryotic cell, wherein the ligand binds to thesingle-polypeptide chain, ligand-dependent, stability-affecting proteinto modulate stability of the fusion protein.
 11. The method of claim 10,wherein the single-polypeptide chain, ligand-dependent,stability-affecting protein is a FKBP variant protein and thenaturally-occurring ligand binding protein is a wild-type FKBP protein.12. The method of claim 10, wherein the protein of interest is TNF-α orIL-2.
 13. The method of claim 10, wherein the introducing of the nucleicacid sequence comprises transforming the eukaryotic cell in culture witha plasmid comprising the nucleic acid to produce stably transformedeukaryotic cells, and implanting the stably transformed eukaryotic cellsinto mice.
 14. The method of claim 10, wherein the introducing thenucleic acid sequence comprises administering a viral vector comprisingthe nucleic acid sequence to an animal that has tumor cells.
 15. Themethod of claim 14, wherein the viral vector is a vaccinia virus.
 16. Amethod for modulating cellular proliferation in an animal, comprising:administering to the animal a nucleic acid comprising a polynucleotidewhich encodes a fusion protein wherein the fusion protein comprises aprotein of interest and a single-polypeptide chain, ligand-dependent,stability-affecting protein derived from a naturally-occurring ligandbinding protein; and administering a ligand which binds preferably tothe single-polypeptide chain, ligand-dependent, stability-affectingprotein as compared to the naturally-occurring ligand binding protein.17. The method of claim 16, wherein the single-polypeptide chain,ligand-dependent, stability-affecting protein is a FKBP variant proteinand the naturally-occurring ligand binding protein is a wild-type FKBPprotein.
 18. The method of claim 16, wherein the protein of interest isTNF-α or IL-2.
 19. The method of claim 16, wherein the administering tothe animal a nucleic acid comprises transforming a eukaryotic cell withthe nucleic acid to produce a stably transformed eukaryotic cell andimplanting the stably transformed eukaryotic tumor cell into the animal.20. The method of claim 16, wherein the animal has tumor cells andwherein the administering to the animal comprises administering a virusto the animal, wherein the virus harbors the nucleic acid.